Formulations of biologics for intravesical instillation

ABSTRACT

Pharmaceutical formulations comprising a clostridial derivative and a permeabilizing agent for intravesical instillation are disclosed.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.14/700,390, filed on Apr. 30, 2015, now U.S. Pat. No. 9,943,576, whichclaims the benefit of U.S. Provisional Application Ser. No. 61/986,346,filed Apr. 30, 2014, and 62/031,302, filed Jul. 31, 2014, allincorporated herein entirely by reference.

FIELD OF INVENTION

The present disclosure relates to pharmaceutical formulations comprisinga clostridial derivative and methods of use thereof. In particular, thepresent disclosure relates to pharmaceutical formulations containing aclostridial derivative for bladder instillation.

BACKGROUND

Neurotoxin therapies, in particular botulinum toxins, have been used intreatments of various medical conditions, including urologicalconditions such as overactive bladder (OAB) and detrusor overactivity.

Botulinum toxin therapy to treat bladder disorders such as overactivebladder (OAB), detrusor overactivity associated with a neurologicalcondition, is typically administered by injection across the urinarybladder wall and into the enervated muscular tissues surrounding thebladder. This approach requires administering about thirty to fortyinjections through the bladder wall, as shown in FIG. 1 . Pharmaceuticaladministration by injection may cause localized pain, and potentiallyexpose patients to blood borne diseases. Among alternativeadministration routes, intravesical instillation allows a drug to bedelivered directly into the bladder by crossing the bladder wall.

The bladder wall is impermeable to most substances. As shown in FIG. 2 ,the stratified urothelium consists of three cellular layers: umbrellacells, intermediate cells, and basal cells. The basal cells are germinalcells that through cell division replace intermediate cells that arepartially differentiated. The highly differentiated and polarizedumbrella cells are located on the lumen of the bladder and are theprimary physical barrier to the movement of substances between the bloodand urine. The apical membrane of the umbrella cells is covered withplaques consisting of proteins called uroplakins and gives the apicalmembrane a thick appearance. The umbrella cells also contain tightjunctions that restrict the paracellular movement of urine and largermolecules through the epithelium.

Thus, there remains a need for pharmaceutical formulations containing aclostridial derivative that can enhance delivery across the urinarybladder wall in lieu of parenteral administration.

Clostridial toxin therapies are successfully used for many indications.Generally, administration of a Clostridial toxin treatment is welltolerated. It has been found that botulinum toxins are able to affectmany different types of neurons in the human body, but for the treatmentof some medical conditions, more specific molecules will beadvantageous. Thus, there remains a need for pharmaceutical formulationscontaining modified clostridial derivatives, such as Targeted ExocytosisModulators (TEMs), targeted for specific types of neuronal cells.

SUMMARY OF THE INVENTION

In some aspects, the present disclosure provides pharmaceuticalformulations for intravesical (urinary bladder) administration,comprising a clostridial derivative and at least one permeabilizingagent, which can permeate the bladder wall of a patient and retain theclostridial derivative's bioactivity to cause a desired therapeuticeffect.

In one aspect, the present disclosure provides a pharmaceuticalcomposition comprising a therapeutically effective amount of aclostridial derivative and at least one permeabilizing agent, whereinthe at least one permeabilizing agent is present in an amount effectiveto substantially and reversibly increase the permeability of the bladderwall to the clostridial derivative.

In another aspect, the present disclosure provides a method for making apharmaceutical formulation suitable for intravesical bladderadministration, the method comprising providing a solution comprising atleast one permeabilizing agent; adding the solution to a compositioncomprising a clostridial derivative. In some embodiments, the methodcomprises adding about 50 ml to about 100 ml of the solution to theclostridial derivative. In some embodiments, the clostridial derivativeis a botulinum toxin. In one embodiment, the method comprises addingabout 50 ml to about 100 ml of an aqueous solution comprising about 1%(w/v) chitosan, analogs or derivatives and about 0.1% (w/v)Triton™X-100, analogs or derivatives, to a botulinum toxin type A. Inone embodiment, the method comprises adding a 50 ml aqueous solutioncomprising 1% (w/v) chitosan and 0.1% (w/v) Triton™X-100 to a vialcontaining about 100 Units or 200 Units of a lyophilized botulinum toxintype A; and mixing gently to rehydrate the lyophilized botulinum toxintype A.

In another aspect, the present pharmaceutical formulation maximizes thebioavailability of the clostridial derivative by preventing orminimizing the adsorption of the clostridial derivative to catheters,deliver device surfaces (syringe, patch, microneedle, engineeredinjector (Bioject, etc.), tubing and containers.

According to another aspect, the present disclosure provides methods fortreating medical disorders in a patient, the methods comprise the stepof administering a pharmaceutical composition provided in accordancewith the present disclosure, thereby treating the medical disorders. Inone embodiment, the present methods alleviate one or more symptoms ofthe medical disorders. In one embodiment, the medical disorders includeneurogenic idiopathic bladder dysfunction, or bladder pain. In someaspects, a method is provided for treating a patient with a neurogenicor idiopathic bladder dysfunction, bladder pain, comprising:intravesically instilling into the bladder of the patient apharmaceutical composition comprising a therapeutically effective amountof a clostridial derivative and at least one permeabilizing agentpresent in an amount effective to substantially increase thepermeability of the bladder wall to the clostridial derivative at atherapeutically effective rate.

Other aspects and variations of the present pharmaceutical formulationsand methods summarized above are also contemplated and will be morefully understood when considered with respect to the followingdisclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the office upon request and paymentof the necessary fee.

FIG. 1 shows a prior art intracystinal injection of a botulinum toxin tothe bladder wall;

FIG. 2 is a schematic of the urothelium, wherein:

-   -   A) The urothelium consists of three cell layers: basal,        intermediate, and superficial umbrella cells.    -   B) The umbrella cells display plaques on their apical membrane        and a cytoplasmic network of vesicles containing fibrils joined        at the tight junction and the desmosomes on the smooth basal        membrane.    -   C) The apical membrane of the urothelium viewed for the lumen        shows the plaque and hinge regions.

FIG. 3 shows a schematic of the human uroepithelial culture model; adiagram of the Transwell® permeable support system with a stratifiedurothelium;

FIG. 4 shows an exemplary graph displaying differential effect ofvarious permeabilizing agents on the permeability of human uroepithelialcells to an exemplary surrogate;

FIGS. 5A and 5B show concentration and time dependent effects of twoexemplary permeabilizing agents on the cell permeability and Light chainactivity of an exemplary TEM surrogate;

FIGS. 6A and 6B show concentration and time dependent effects of twoexemplary permeabilizing agents on the cell permeability and Light chainactivity of a botulinum toxin type A;

FIG. 7 shows the effect of an exemplary permeabilizing agent on the cellpermeability and Light chain activity of a botulinum toxin type A at pH6.0 or 8.0;

FIGS. 8A-10 display data obtained in vivo, wherein effect of exemplaryformulations in accordance of the present disclosure was quantified inrats;

FIG. 8A is a pictorial diagram establishing a basis for assessing ratbladder integrity, using an ordinal scoring method of 0-5; ‘0’ being anormal bladder and ‘5’ showing severe pathology;

FIG. 8B is a pictorial diagram showing a basis for assessing thepenetration of an exemplary surrogate into the bladder wall. Theassessment relies on the extent of SNAP25-197 staining corresponding toVAChT staining of parasympathetic nerve fibers in rat bladder using anordinal scoring method of 0-5; ‘0’ being no SNAP25-197 staining detectedand ‘4.5’ showing near complete overlap of both biomarkers. A score of‘5’ was never observed;

FIG. 9A shows the effect of an exemplary formulation comprising anexemplary surrogate in accordance with aspect of the present disclosurefollowing instillation into normal bladders, as measured by: (1) theextent of penetration of the surrogate at various concentrations ofexemplary permeabilizing agents; and (2) bladder tissue integrity. Theextent of penetration was correlated to the extent of SNAP25-197staining (blue bars, or first bar of two contiguous bars) and thebladder tissue damage was based on H&E staining (red bars, or second barof two contiguous bars). The bars without an adjoining bar show extentof SNAP25-197 staining.

FIG. 9B shows the effect of the exemplary formulation followinginstillation into bladders from Interstitial cystitis (IC) model, asmeasured by: (1) the extent of penetration of an exemplary surrogate atvarious concentrations of exemplary permeabilizing agents; and (2)bladder tissue integrity. The extent of penetration was correlated tothe extent of SNAP25-197 staining (blue bars, or first bar of twocontiguous bars) and the bladder tissue damage was based on H&E staining(red bars, or second bar of two contiguous bars);

FIG. 10 shows the effect of an exemplary formulation comprising abotulinum toxin complex (20 U) following instillation into normalbladders at pH 6.0 or 8.0, as measured by: (1) the extent of penetrationof the botulinum toxin complex at various concentrations of an exemplarypermeabilizing agent; and (2) bladder tissue integrity. The extent ofpenetration was correlated to the extent of SNAP25-197 staining (bluebars, or first bar of two contiguous bars) and the bladder tissue damagewas based on H&E staining (red bars, or second bar of two contiguousbars); and

FIG. 11 shows the effect of an exemplary permeabilizing agent on themucoadhesion of a surrogate.

FIG. 12 is a pictorial diagram establishing a basis for assessing theextent of penetration into the bladder of a test formulation. The extentof penetration was correlated to the extent of SNAP25-197 staining;

FIG. 13 is a pictorial diagram showing exemplary immunohistochemistryresults from two bladders having IHC scores of “4” and “0”;

FIGS. 14A-D are illustrative photomicrographs showing different statesof the bladder tissue after instillation; and

FIG. 15 is a graph displaying the immunohistochemistry (IHC) scores ofsome exemplary formulations according to aspects of the presentdisclosure.

DESCRIPTION

Botulinum neurotoxins (BoNTs), for example, BoNT/A, BoNT/B, etc., act onthe nervous system by blocking the release of neurosecretory substancesincluding neurotransmitters. The action of BoNT is initiated by itsbinding to a receptor molecule on the cell surface. The resultingtoxin-receptor complex then undergoes endocytosis. Once inside the cell,BoNT cleaves exocytotic specific proteins responsible forneurotransmitter docking and release from the cell known as the SNAREproteins (soluble N-ethylmaleimide-sensitive factor attachment proteinreceptor). The resulting transient chemodenervation has been utilizedmedically to block motor neurotransmission at the neuromuscularjunction, leading to a variety of therapeutic applications.

Aspects of the present disclosure provide, in part, a pharmaceuticalformulation suitable for intravesical bladder delivery, comprising aclostridial derivative and at least one permeabilizing agent.

In one aspect, the present disclosure provides in part a pharmaceuticalcomposition comprising a therapeutically effective amount of aclostridial derivative and at least one permeabilizing agent, whereinthe at least one permeabilizing agent is present in an amount effectiveto substantially and reversibly increase the permeability of the bladderwall to the clostridial derivative. In some embodiments, the clostridialderivative is a botulinum toxin. In some embodiments, the at least onepermeabilizing agent comprises a surfactant and a mucoadhesive.

Definitions

As used herein, the words or terms set forth below have the followingdefinitions:

“About” or “approximately” as used herein means within an acceptableerror range for the particular value as determined by one of ordinaryskill in the art, which will depend in part on how the value is measuredor determined, (i.e., the limitations of the measurement system). Forexample, “about” can mean within 1 or more than 1 standard deviations,per practice in the art. Where particular values are described in theapplication and claims, unless otherwise stated, the term “about” meanswithin an acceptable error range for the particular value.

“Active pharmaceutical ingredient” (API) means an ingredient that exertsan effect upon or after administration to a subject or patient. API'scan include, for example, a native or recombinant, clostridialneurotoxin, e.g. a botulinum toxin, recombinant modified toxins,fragments thereof, TEMs, and combinations thereof.

“Administration”, or “to administer” means the step of giving (i.e.administering) a pharmaceutical composition to a subject, oralternatively a subject receiving a pharmaceutical composition. Thepharmaceutical compositions disclosed herein can be locally administeredby various methods. For example, intramuscular, intradermal,subcutaneous administration, intrathecal administration, intraperitonealadministration, topical (transdermal), instillation, and implantation(for example, of a slow-release device such as polymeric implant orminiosmotic pump) can all be appropriate routes of administration.

“Alleviating” means a reduction in the occurrence of a pain, of aheadache, of a hyperactive muscle, or of any symptom or cause of acondition or disorder. Thus, alleviating includes some reduction,significant reduction, near total reduction, and total reduction.

“Animal protein free” means the absence of blood derived, blood pooledand other animal derived products or compounds. “Animal” means a mammal(such as a human), bird, reptile, fish, insect, spider or other animalspecies. “Animal” excludes microorganisms, such as bacteria. Thus, ananimal protein free pharmaceutical composition can include a botulinumneurotoxin, a recombinant modified toxin, or a TEM. For example, an“animal protein free” pharmaceutical composition means a pharmaceuticalcomposition which is either substantially free or essentially free orentirely free of a serum derived albumin, gelatin and other animalderived proteins, such as immunoglobulins. An example of an animalprotein free pharmaceutical composition is a pharmaceutical compositionwhich comprises or which consists of a botulinum toxin, a TEM, or arecombinant modified toxin (as the active ingredient) and a suitablepolysaccharide as a stabilizer or excipient.

“Biological activity” describes the beneficial or adverse effects of adrug on living matter. When a drug is a complex chemical mixture, thisactivity is exerted by the substance's active ingredient but can bemodified by the other constituents. Biological activity can be assessedas potency or as toxicity by an in vivo LD₅₀ or ED₅₀ assay, or throughan in vitro assay such as, for example, cell-based potency assays asdescribed in U.S. publications 20100203559, 20100233802, 20100233741 andU.S. Pat. No. 8,198,034, each of which is hereby incorporated byreference in its entirety.

“Botulinum toxin” means a neurotoxin produced by Clostridium botulinum,as well as a botulinum toxin (or the light chain or the heavy chainthereof) made recombinantly by a non-Clostridial species. The phrase“botulinum toxin”, as used herein, encompasses the botulinum toxinserotypes A, B, C, D, E, F and G, and their subtypes and any other typesof subtypes thereof, or any re-engineered proteins, analogs,derivatives, homologs, parts, sub-parts, variants, or versions, in eachcase, of any of the foregoing. “Botulinum toxin”, as used herein, alsoencompasses a “modified botulinum toxin”. Further “botulinum toxin” asused herein also encompasses a botulinum toxin complex, (for example,the 300, 500 and 900 kDa complexes), as well as the neurotoxic componentof the botulinum toxin (150 kDa) that is unassociated with the complexproteins.

“Clostridial derivative” refers to a molecule which contains any part ofa clostridial toxin. As used herein, the term “clostridial derivative”encompasses native or recombinant neurotoxins, recombinant modifiedtoxins, fragments thereof, a Targeted vesicular Exocytosis Modulator(TEM), or combinations thereof.

“Clostridial toxin” refers to any toxin produced by a Clostridial toxinstrain that can execute the overall cellular mechanism whereby aClostridial toxin intoxicates a cell and encompasses the binding of aClostridial toxin to a low or high affinity Clostridial toxin receptor,the internalization of the toxin/receptor complex, the translocation ofthe Clostridial toxin light chain into the cytoplasm and the enzymaticmodification of a Clostridial toxin substrate. Non-limiting examples ofClostridial toxins include a Botulinum toxin like BoNT/A, a BoNT/B, aBoNT/C₁, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a Tetanus toxin (TeNT),a Baratii toxin (BaNT), and a Butyricum toxin (BuNT). The BoNT/C₂cytotoxin and BoNT/C₃ cytotoxin, not being neurotoxins, are excludedfrom the term “Clostridial toxin.” A Clostridial toxin disclosed hereinincludes, without limitation, naturally occurring Clostridial toxinvariants, such as, e.g., Clostridial toxin isoforms and Clostridialtoxin subtypes; non-naturally occurring Clostridial toxin variants, suchas, e.g., conservative Clostridial toxin variants, non-conservativeClostridial toxin variants, Clostridial toxin chimeric variants andactive Clostridial toxin fragments thereof, or any combination thereof.A Clostridial toxin disclosed herein also includes a Clostridial toxincomplex. As used herein, the term “Clostridial toxin complex” refers toa complex comprising a Clostridial toxin and non-toxin associatedproteins (NAPs), such as, e.g., a Botulinum toxin complex, a Tetanustoxin complex, a Baratii toxin complex, and a Butyricum toxin complex.Non-limiting examples of Clostridial toxin complexes include thoseproduced by a Clostridium botulinum, such as, e.g., a 900-kDa BoNT/Acomplex, a 500-kDa BoNT/A complex, a 300-kDa BoNT/A complex, a 500-kDaBoNT/B complex, a 500-kDa BoNT/C₁ complex, a 500-kDa BoNT/D complex, a300-kDa BoNT/D complex, a 300-kDa BoNT/E complex, and a 300-kDa BoNT/Fcomplex.

“″Effective amount” as applied to the biologically active ingredientmeans that amount of the ingredient which is generally sufficient toinduce a desired change in the subject. For example, where the desiredeffect is a reduction in an autoimmune disorder symptom, an effectiveamount of the ingredient is that amount which causes at least asubstantial reduction of the autoimmune disorder symptom, and withoutresulting in significant toxicity.

“Effective amount” as applied to a non-active ingredient constituent ofa pharmaceutical composition (such as a stabilizer used for mixing witha botulinum toxin) refers to that amount of the non-active ingredientconstituent which is sufficient to positively influence the releaseand/or activity of the active ingredient when administered to anindividual. This “effective amount” can be determined based on theteaching in this specification and the general knowledge in the art.

“Entirely free (i.e. “consisting of” terminology) means that within thedetection range of the instrument or process being used, the substancecannot be detected or its presence cannot be confirmed.

“Essentially free” (or “consisting essentially of”) means that onlytrace amounts of the substance can be detected.

“Light chain” means the light chain of a clostridial neurotoxin. It hasa molecular weight of about 50 kDa, and can be referred to as the Lchain, L, or as the proteolytic domain (amino acid sequence) of abotulinum neurotoxin.

“Heavy chain” means the heavy chain of a botulinum neurotoxin. It has amolecular weight of about 100 kDa and can be referred to as the H chain,or as H.

H_(C) means a fragment (about 50 kDa) derived from the H chain of abotulinum neurotoxin which is approximately equivalent to the carboxylend segment of the H chain, or the portion corresponding to thatfragment in the intact H chain. It is believed to contain the portion ofthe natural or wild type botulinum neurotoxin involved in high affinity,presynaptic binding to motor neurons.

H_(N) means a fragment (about 50 kDa) derived from the H chain of abotulinum neurotoxin which is approximately equivalent to the amino endsegment of the H chain, or a portion corresponding to that fragment. Itis believed to contain the portion of the natural or wild type botulinumneurotoxin involved in the translocation of the L chain across anintracellular endosomal membrane.

LH_(N) or L-H_(N) means a fragment derived from a clostridial neurotoxinthat contains the L chain, or a functional fragment thereof coupled tothe H_(N) domain. It can be obtained from the intact clostridialneurotoxin by proteolysis, so as to remove or to modify the H_(C)domain.

“Implant” means a controlled release (e.g., pulsatile or continuous)composition or drug delivery system. The implant can be, for example,injected, inserted or implanted into a human body.

“Intravesical administration” refers to the injection of a givensubstance directly into the bladder via a urethral catheter.

“Local administration” means direct administration of a pharmaceuticalat or to the vicinity of a site on or within an animal body, at whichsite a biological effect of the pharmaceutical is desired, such as via,for example, intramuscular or intra- or subdermal injection or topicaladministration. Local administration excludes systemic routes ofadministration, such as intravenous or oral administration. Topicaladministration is a type of local administration in which apharmaceutical agent is applied to a patient's skin.

“Modified botulinum toxin” means a botulinum toxin that has had at leastone of its amino acids deleted, modified, or replaced, as compared to anative botulinum toxin. Additionally, the modified botulinum toxin canbe a recombinantly produced neurotoxin, or a derivative or fragment of arecombinantly made neurotoxin. A modified botulinum toxin retains atleast one biological activity of the native botulinum toxin, such as,the ability to bind to a botulinum toxin receptor, or the ability toinhibit neurotransmitter release from a neuron. One example of amodified botulinum toxin is a botulinum toxin that has a light chainfrom one botulinum toxin serotype (such as serotype A), and a heavychain from a different botulinum toxin serotype (such as serotype B).Another example of a modified botulinum toxin is a botulinum toxincoupled to a neurotransmitter, such as substance P.

“Mutation” means a structural modification of a naturally occurringprotein or nucleic acid sequence. For example, in the case of nucleicacid mutations, a mutation can be a deletion, addition or substitutionof one or more nucleotides in the DNA sequence. In the case of a proteinsequence mutation, the mutation can be a deletion, addition orsubstitution of one or more amino acids in a protein sequence. Forexample, a specific amino acid comprising a protein sequence can besubstituted for another amino acid, for example, an amino acid selectedfrom a group which includes the amino acids alanine, asparagine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, tyrosine or any othernatural or non-naturally occurring amino acid or chemically modifiedamino acids. Mutations to a protein sequence can be the result ofmutations to DNA sequences that when transcribed, and the resulting mRNAtranslated, produce the mutated protein sequence. Mutations to a proteinsequence can also be created by fusing a peptide sequence containing thedesired mutation to a desired protein sequence.

“Patient” means a human or non-human subject receiving medical orveterinary care. Accordingly, as disclosed herein, the compositions andmethods can be used in treating any animal, such as, for example,mammals, or the like.

“Peripherally administering” or “peripheral administration” meanssubdermal, intradermal, transdermal, or subcutaneous administration, butexcludes intramuscular administration. “Peripheral” means in a subdermallocation, and excludes visceral sites.

“Permeabilizing agent” refers to any naturally occurring or syntheticcompound, substance or molecule which has the ability to enhance thepermeability of a surface, including but not limited to the skin, thebladder wall, and the like, to a selected compound, such as an API(Active pharmaceutical ingredient).

“Permeation-effective amount” refers to an amount effective tosubstantially increase the permeability of a surface to a therapeuticagent at a therapeutically effective rate. For intravesical bladderdelivery, a permeation-effective amount refers to an amount sufficientto substantially increase the permeability of the bladder wall to aclostridial derivative for a desired time interval without irreversiblydamaging the bladder wall, after which time the original selectiveimpermeability of the bladder wall may be restored.

“Pharmaceutical composition” means a composition comprising an activepharmaceutical ingredient, such as, for example, a botulinum toxin, andat least one additional ingredient, such as, for example, a stabilizeror excipient or the like. A pharmaceutical composition is therefore aformulation which is suitable for diagnostic or therapeuticadministration to a subject, such as a human patient. The pharmaceuticalcomposition can be, for example, in a lyophilized or vacuum driedcondition, a solution formed after reconstitution of the lyophilized orvacuum dried pharmaceutical composition, or as a solution or solid whichdoes not require reconstitution.

The constituent ingredients of a pharmaceutical composition can beincluded in a single composition (that is, all the constituentingredients, except for any required reconstitution fluid, are presentat the time of initial compounding of the pharmaceutical composition) oras a two-component system, for example a vacuum-dried compositionreconstituted with a reconstitution vehicle which can, for example,contain an ingredient not present in the initial compounding of thepharmaceutical composition. A two-component system can provide severalbenefits, including that of allowing incorporation of ingredients whichare not sufficiently compatible for long-term shelf storage with thefirst component of the two component system. For example, thereconstitution vehicle may include a preservative which providessufficient protection against microbial growth for the use period, forexample one-week of refrigerated storage, but is not present during thetwo-year freezer storage period during which time it might degrade thetoxin. Other ingredients, which may not be compatible with a botulinumtoxin or other ingredients for long periods of time, can be incorporatedin this manner; that is, added in a second vehicle (e.g. in thereconstitution vehicle) at the approximate time of use. A pharmaceuticalcomposition can also include preservative agents such as benzyl alcohol,benzoic acid, phenol, parabens and sorbic acid. Pharmaceuticalcompositions can include, for example, excipients, such as surfaceactive agents; dispersing agents; inert diluents; granulating anddisintegrating agents; binding agents; lubricating agents;preservatives; physiologically degradable compositions such as gelatin;aqueous vehicles and solvents; oily vehicles and solvents; suspendingagents; dispersing or wetting agents; emulsifying agents, demulcents;buffers; salts; thickening agents; fillers; antioxidants; stabilizingagents; and pharmaceutically acceptable polymeric or hydrophobicmaterials and other ingredients known in the art and described, forexample in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa., which is incorporated herein by reference.

“Recombinant modified toxin” means a recombinant toxin that shares someor most of the domains with a Botulinum toxin but may or may not targetthe same cells as native Botulinum neurotoxin.

“Stabilizing”, “stabilizes”, or “stabilization” means the retention ofat least about 20% of the biological activity of an activepharmaceutical ingredient (“API”) that has been reconstituted, whencompared to the API prior to reconstitution. For example, upon (1)preparation of serial dilutions from a bulk or stock solution, or (2)upon reconstitution of a lyophilized, or vacuum dried botulinum toxincontaining pharmaceutical composition which has been stored at or belowabout −2° C. for between six months and four years, or (3) for anaqueous solution botulinum toxin containing pharmaceutical compositionwhich has been stored at between about 2° C. and about 8° C. for fromsix months to four years, the botulinum toxin present in thereconstituted or aqueous solution pharmaceutical composition has (in thepresence of a compound which is stabilizing, stabilizes or whichprovides stabilization to the API) greater than about 20% and up toabout 100% of the potency or toxicity that the biologically activebotulinum toxin had prior to being incorporated into the pharmaceuticalcomposition.

“Stabilizing agent”, “stabilization agent” or “stabilizer” means asubstance that acts to stabilize an API such that the potency of thepharmaceutical composition is increased relative to an unstabilizedcomposition.

“Stabilizers” can include excipients, and can include protein andnon-protein molecules.

“Substantially free” means present at a level of less than one percentby weight of the pharmaceutical composition.

“TEM” as used herein, is synonymous with “Targeted Exocytosis Modulator”or “retargeted endopeptidase.” Because of its numerous characteristics,“TEM” will be disclosed in further details at the end of the“Definition” section.

“Therapeutic formulation” means a formulation can be used to treat andthereby alleviate a disorder or a disease, such as, for example, adisorder or a disease characterized by hyperactivity (i.e. spasticity)of a peripheral muscle.

“Therapeutically effective amount” refers to an amount sufficient toachieve a desired therapeutic effect.

“Topical administration” excludes systemic administration of theneurotoxin. In other words, and unlike conventional therapeutictransdermal methods, topical administration of botulinum toxin does notresult in significant amounts, such as the majority of, the neurotoxinpassing into the circulatory system of the patient.

“Treating” means to alleviate (or to eliminate) at least one symptom ofa condition or disorder, such as, for example, wrinkles, spasticity,depression, pain (such as, for example, headache pain), bladderoveractivity, or the like, either temporarily or permanently.

“Variant” means a clostridial neurotoxin, such as wild-type botulinumtoxin serotype A, B, C, D, E, F or G, that has been modified by thereplacement, modification, addition or deletion of at least one aminoacid relative to wild-type botulinum toxin, which is recognized by atarget cell, internalized by the target cell, and catalytically cleavesa SNARE (SNAP (Soluble NSF Attachment Protein) Receptor) protein in thetarget cell.

An example of a variant neurotoxin component can comprise a variantlight chain of a botulinum toxin having one or more amino acidssubstituted, modified, deleted and/or added. This variant light chainmay have the same or better ability to prevent exocytosis, for example,the release of neurotransmitter vesicles. Additionally, the biologicaleffect of a variant may be decreased compared to the parent chemicalentity. For example, a variant light chain of a botulinum toxin type Ahaving an amino acid sequence removed may have a shorter biologicalpersistence than that of the parent (or native) botulinum toxin type Alight chain.

“Vehicle” or “reconstitution vehicle” means a liquid composition thatcan be used to reconstitute a solid botulinum formulation into a liquidbotulinum pharmaceutical composition.

“Wild type neuronal binding moiety” means that portion of a neurotoxinwhich is native to the neurotoxin and which exhibits a specific bindingaffinity for a receptor on a neuron. Thus, wild type or native neuronalbinding moiety excludes a binding moiety with is not native to theneurotoxin.

TEMs

Generally, a TEM comprises an enzymatic domain from a Clostridial toxinlight chain, a translocation domain from a Clostridial toxin heavychain, and a targeting domain. The targeting domain of a TEM provides analtered cell targeting capability that targets the molecule to areceptor other than the native Clostridial toxin receptor utilized by anaturally-occurring Clostridial toxin. This re-targeted capability isachieved by replacing the naturally-occurring binding domain of aClostridial toxin with a targeting domain having a binding activity fora non-Clostridial toxin receptor. Although binding to a non-Clostridialtoxin receptor, a TEM undergoes all the other steps of the intoxicationprocess including internalization of the TEM/receptor complex into thecytoplasm, formation of the pore in the vesicle membrane and di-chainmolecule, translocation of the enzymatic domain into the cytoplasm, andexerting a proteolytic effect on a component of the SNARE complex of thetarget cell.

As used herein, the term “Clostridial toxin enzymatic domain” refers toa Clostridial toxin polypeptide located in the light chain of aClostridial toxin that executes the enzymatic target modification stepof the intoxication process. A Clostridial toxin enzymatic domainincludes a metalloprotease region containing a zinc-dependentendopeptidase activity which specifically targets core components of theneurotransmitter release apparatus. Thus, a Clostridial toxin enzymaticdomain specifically targets and proteolytically cleavages of aClostridial toxin substrate, such as, e.g., SNARE proteins like aSNAP-25 substrate, a VAMP substrate and a Syntaxin substrate.

A Clostridial toxin enzymatic domain includes, without limitation,naturally occurring Clostridial toxin enzymatic domain variants, suchas, e.g., Clostridial toxin enzymatic domain isoforms and Clostridialtoxin enzymatic domain subtypes; non-naturally occurring Clostridialtoxin enzymatic domain variants, such as, e.g., conservative Clostridialtoxin enzymatic domain variants, non-conservative Clostridial toxinenzymatic domain variants, Clostridial toxin enzymatic domain chimeras,active Clostridial toxin enzymatic domain fragments thereof, or anycombination thereof. Non-limiting examples of a Clostridial toxinenzymatic domain include, e.g., a BoNT/A enzymatic domain, a BoNT/Benzymatic domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain,a BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G enzymaticdomain, a TeNT enzymatic domain, a BaNT enzymatic domain, and a BuNTenzymatic domain.

As used herein, the term “Clostridial toxin translocation domain” refersto a Clostridial toxin polypeptide located within the amino-terminalhalf of the heavy chain of a Clostridial toxin that executes thetranslocation step of the intoxication process. The translocation stepappears to involve an allosteric conformational change of thetranslocation domain caused by a decrease in pH within the intracellularvesicle. This conformational change results in the formation of a porein the vesicular membrane that permits the movement of the light chainfrom within the vesicle into the cytoplasm. Thus, a Clostridial toxintranslocation domain facilitates the movement of a Clostridial toxinlight chain across a membrane of an intracellular vesicle into thecytoplasm of a cell.

A Clostridial toxin translocation domain includes, without limitation,naturally occurring Clostridial toxin translocation domain variants,such as, e.g., Clostridial toxin translocation domain isoforms andClostridial toxin translocation domain subtypes; non-naturally occurringClostridial toxin translocation domain variants, such as, e.g.,conservative Clostridial toxin translocation domain variants,non-conservative Clostridial toxin translocation domain variants,Clostridial toxin translocation domain chimerics, active Clostridialtoxin translocation domain fragments thereof, or any combinationthereof. Non-limiting examples of a Clostridial toxin translocationdomain include, e.g., a BoNT/A translocation domain, a BoNT/Btranslocation domain, a BoNT/C1 translocation domain, a BoNT/Dtranslocation domain, a BoNT/E translocation domain, a BoNT/Ftranslocation domain, a BoNT/G translocation domain, a TeNTtranslocation domain, a BaNT translocation domain, and a BuNTtranslocation domain.

As used herein, the term “targeting domain” is synonymous with “bindingdomain” or “targeting moiety” and refers to a peptide or polypeptidethat executes the receptor binding and/or complex internalization stepsof the intoxication process, with the proviso that the binding domain isnot identical to a Clostridial toxin binding domain found within thecarboxyl-terminal half of the heavy chain of a Clostridial toxin. Atargeting domain includes a receptor binding region that confers thebinding activity and/or specificity of the targeting domain for itscognate receptor. As used herein, the term “cognate receptor” refers toa receptor for which the targeting domain preferentially interacts withunder physiological conditions, or under in vitro conditionssubstantially approximating physiological conditions. As used herein,the term “preferentially interacts” is synonymous with “preferentiallybinding” and refers to an interaction that is statisticallysignificantly greater in degree relative to a control. With reference toa targeting domain disclosed herein, a targeting domain binds to itscognate receptor to a statistically significantly greater degreerelative to a non-cognate receptor. Said another way, there is adiscriminatory binding of the targeting domain to its cognate receptorrelative to a non-cognate receptor. Thus, a targeting domain directsbinding to a TEM-specific receptor located on the plasma membranesurface of a target cell.

A targeting domain disclosed herein may be one that preferentiallyinteracts with a receptor located on a sensory neuron. In anotherembodiment, a targeting domain disclosed herein may be one thatpreferentially interacts with a receptor located on a sympatheticneuron, or a parasympathetic neuron.

In another embodiment, a targeting domain disclosed herein is an opioidpeptide targeting domain, a galanin peptide targeting domain, a PARpeptide targeting domain, a somatostatin peptide targeting domain, aneurotensin peptide targeting domain, a SLURP peptide targeting domain,an angiotensin peptide targeting domain, a tachykinin peptide targetingdomain, a Neuropeptide Y related peptide targeting domain, a kininpeptide targeting domain, a melanocortin peptide targeting domain, or agranin peptide targeting domain, a glucagon like hormone peptidetargeting domain, a secretin peptide targeting domain, a pituitaryadenylate cyclase activating peptide (PACAP) peptide targeting domain, agrowth hormone-releasing hormone (GHRH) peptide targeting domain, avasoactive intestinal peptide (VIP) peptide targeting domain, a gastricinhibitory peptide (GIP) peptide targeting domain, a calcitonin peptidetargeting domain, a visceral gut peptide targeting domain, aneurotrophin peptide targeting domain, a head activator (HA) peptide, aglial cell line-derived neurotrophic factor (GDNF) family of ligands(GFL) peptide targeting domain, a RF-amide related peptide (RFRP)peptide targeting domain, a neurohormone peptide targeting domain, or aneuroregulatory cytokine peptide targeting domain, an interleukin (IL)targeting domain, vascular endothelial growth factor (VEGF) targetingdomain, an insulin-like growth factor (IGF) targeting domain, anepidermal growth factor (EGF) targeting domain, a Transformation GrowthFactor-β (TGFβ) targeting domain, a Bone Morphogenetic Protein (BMP)targeting domain, a Growth and Differentiation Factor (GDF) targetingdomain, an activin targeting domain, or a Fibroblast Growth Factor (FGF)targeting domain, or a Platelet-Derived Growth Factor (PDGF) targetingdomain.

An opioid peptide targeting domain may include an enkephalin peptide, abovine adrenomedullary-22 (BAM22) peptide, an endomorphin peptide, anendorphin peptide, a dynorphin peptide, a nociceptin peptide, or ahemorphin peptide.

Thus, a TEM can comprise a targeting domain in any and all locationswith the proviso that TEM is capable of performing the intoxicationprocess. Non-limiting examples include, locating a targeting domain atthe amino terminus of a TEM; locating a targeting domain between aClostridial toxin enzymatic domain and a Clostridial toxin translocationdomain of a TEM; and locating a targeting domain at the carboxylterminus of a TEM. Other non-limiting examples include, locating atargeting domain between a Clostridial toxin enzymatic domain and aClostridial toxin translocation domain of a TEM. The enzymatic domain ofnaturally-occurring Clostridial toxins contains the native startmethionine. Thus, in domain organizations where the enzymatic domain isnot in the amino-terminal location an amino acid sequence comprising thestart methionine should be placed in front of the amino-terminal domain.Likewise, where a targeting domain is in the amino-terminal position, anamino acid sequence comprising a start methionine and a proteasecleavage site may be operably-linked in situations in which a targetingdomain requires a free amino terminus, see, e.g., Shengwen Li et al.,Degradable Clostridial Toxins, U.S. patent application Ser. No.11/572,512 (Jan. 23, 2007), which is hereby incorporated by reference inits entirety. In addition, it is known in the art that when adding apolypeptide that is operably-linked to the amino terminus of anotherpolypeptide comprising the start methionine that the original methionineresidue can be deleted.

A TEM disclosed herein may optionally comprise an exogenous proteasecleavage site that allows the use of an exogenous protease to convertthe single-chain polypeptide form of a TEM into its more active di-chainform. As used herein, the term “exogenous protease cleavage site” issynonymous with a “non-naturally occurring protease cleavage site” or“non-native protease cleavage site” and means a protease cleavage sitethat is not naturally found in a di-chain loop region from a naturallyoccurring Clostridial toxin.

Although TEMs vary in their overall molecular weight because the size ofthe targeting domain, the activation process and its reliance on anexogenous cleavage site is essentially the same as that forrecombinantly-produced Clostridial toxins. See e.g., Steward, et al.,Activatable Clostridial Toxins, US 2009/0081730; Steward, et al.,Modified Clostridial Toxins with Enhanced Translocation Capabilities andAltered Targeting Activity For Non-Clostridial Toxin Target Cells, U.S.patent application Ser. No. 11/776,075; Steward, et al., ModifiedClostridial Toxins with Enhanced Translocation Capabilities and AlteredTargeting Activity for Clostridial Toxin Target Cells, US 2008/0241881;Steward, et al., Degradable Clostridial Toxins, US 2011/0287517, each ofwhich is hereby incorporated by reference. In general, the activationprocess that converts the single-chain polypeptide into its di-chainform using exogenous proteases can be used to process TEMs having atargeting domain organized in an amino presentation, centralpresentation, or carboxyl presentation arrangement. This is because formost targeting domains the amino-terminus of the moiety does notparticipate in receptor binding. As such, a wide range of proteasecleavage sites can be used to produce an active di-chain form of a TEM.However, targeting domains requiring a free amino-terminus for receptorbinding require a protease cleavage site whose scissile bond is locatedat the carboxyl terminus. The use of protease cleavage site in thedesign of a TEM are described in, e.g., Steward, et al., ActivatableClostridial toxins, US 2009/0069238; Ghanshani, et al., ModifiedClostridial Toxins Comprising an Integrated Protease CleavageSite-Binding Domain, US 2011/0189162; and Ghanshani, et al., Methods ofIntracellular Conversion of Single-Chain Proteins into their Di-chainForm, International Patent Application Serial No. PCT/US2011/22272, eachof which is incorporated by reference in its entirety.

Pharmaceutical Compositions

Aspects of the present disclosure provide, in part, a pharmaceuticalcomposition suitable for intravesical bladder delivery, comprising aclostridial derivative and at least one permeabilizing agent.

Clostridial Derivative:

A clostridial derivative as defined herein encompasses native orrecombinant neurotoxins, recombinant modified toxins, fragments thereof,a Targeted vesicular Exocytosis Modulator (TEM), or combinationsthereof. In some embodiments, the clostridial derivative is a native ormodified botulinum toxin. In one embodiment, the clostridial derivativeis a botulinum toxin type A. In some embodiments, the clostridialderivative is a botulinum type B, C₁, D, E or F. In alternativeembodiments, the clostridial derivative comprises a TEM.

In some embodiments, the present pharmaceutical composition comprises atherapeutically effective amount of the clostridial derivative. Atherapeutically effective amount refers to the total amount of theclostridial derivative administered to an individual in one setting. Assuch, an effective amount of a Clostridial derivative and/or TEM doesnot refer to the amount administered per site. For example, an effectiveamount of a Clostridial toxin, such as a botulinum toxin, administeredto an individual may be 10 U, whereas the amount of toxin administeredper site may be 2 U, i.e., 2 U at five different sites. In someembodiments, the therapeutically effective amount of the botulinum toxinranges from 10 U to 1000 U, more preferably from about 50 U to about 500U.

With reference to a combination therapy comprising a Clostridial toxinand a TEM, an effective amount of a Clostridial toxin is one where incombination with a TEM the amount of the Clostridial toxin achieves thedesired therapeutic effect, but such an amount administered on its ownwould be ineffective. For example, typically about 75-125 U of BOTOX®(Allergan, Inc., Irvine, Calif.), a BoNT/A, is administered byintramuscular injection per muscle undergoing dystonic spasms in orderto treat cervical dystonia. In combination therapy, a suboptimaleffective amount of BoNT/A would be administered to treat cervicaldystonia when such toxin is used in a combined therapy with a TEM.

Permeabilizing Agents

Examples of permeabilizing agents include but are not limited to anionicsurfactants, cationic surfactants, nonionic surfactants, glycols,chelators, cationic polymers, mucoadhesives, polypeptides, or mixturesthereof.

In some embodiments, the permeabilizing agent selectively binds to theclostridial derivative, such as a botulinum toxin, to form a complex. Inalternative embodiments, the permeabilizing agent does not bind to thebotulinum toxin. In alternative embodiments, the permeabilizing agentinteracts with the clostridial derivative through Van der Wallinteractions.

In certain embodiments, the permeabilizing agent can be anionicsurfactants. Examples of anionic surfactants suitable for the presentformulation include but are not limited to SDS, sodium lauryl sulfate,their analogs, derivatives or any combinations thereof.

In certain embodiments, the permeabilizing agent can be cationicsurfactants. Examples of cationic surfactants suitable for the presentformulation include but are not limited to Protamine sulfate,benzalkonium bromide, quaternary ammonium salts such as poly(dimethylimino)-2-butene-1, 4-diyl chloride, α-[4-tris (2-hydroxyethyl)ammonium-2-butenyl-w-tris (2-hydroxyethyl) ammonium]-dichloride(chemical registry number 75345-27-6) generally available aspolyquaternium 1® from ONYX Corporation, benzalkonium halides, andbiguanides such as salts of alexidine, alexidine free base, salts ofchlorhexidine, hexamethylene biguanides and their polymers, analogs andderivatives. The salts of alexidine and chlorhexidine can be eitherorganic or inorganic and are typically nitrates, acetates, phosphates,sulfates, halides and the like, or any combination thereof, and thelike.

In certain embodiments, the permeabilizing agent can be other cationicagents, including but not limited to poly(ethylenimine) (PEI),Oleylamine, dioleyl-phosphatidylethanolamine (DOPE) and

dioleoyl-trimethylammonium-propane (DOTAP), their analogs, derivatives,or combinations thereof.

In certain embodiments, the permeabilizing agent can comprisepolyethylene glycol (PEG) or polyethylene oxide (PEO). The PEG cancomprise, for example, PEG with a molecular mass ranging from about 200grams per mole (g/m) to about 20,000 grams per mole (g/m). In oneembodiment, the permeabilizing agent comprises Polyethylene glycol 3350.

In certain embodiments the permeabilizing agent can comprise apoloxamer. The poloxamer can comprise, for example, P80, P124, P188,P237, P338, and P407, their analogs, derivatives or combinationsthereof. In certain embodiments the permeabilizing agent can comprise apovidone (PVP). The PVP can comprise, for example, PVP polymers, andanalogs or derivatives.

In certain embodiments, the permeabilizing agent can comprise L or Dpolypeptides, of molecular weight ranging from about 1000 to about100000 daltons. In one embodiment, the permeabilizing agent isPoly-L-lysine. In another embodiment, the permeabilizing agent includescell-penetrating peptides.

In certain embodiments, the permeabilizing agent can comprise benzylalcohol and the like, Polyhexamethylene Biguanide (high and/or lowmolecular weight) and the like, proteins including but not limitednative or recombinant human sera albumin, polymyxin B and the like, ormixtures thereof.

In certain embodiments, the permeabilizing agent includes nonionicsurfactants. Examples of nonionic surfactants suitable for the presentformulation include but are not limited to alkyl aryl polyethers andanalogs, derivatives thereof (e.g. TX-100, analogs or derivatives),poloxamers, polyoxyethylene ethers (e.g. Brij families), Tween, BigCHAPS, Deoxy Big CHAPS, Tyloxapol, Sorbitan monooleate (SPAN) 20, 40,60, Cremophor EL, Alpha-Tocopherol TPGS, Polyoxyl stearate 40, analogsand derivatives, or combinations thereof.

In some embodiments, the permeabilizing agent comprises an alkyl arylpolyether, analogs or derivatives. In one embodiment, the permeabilizingagent comprises octyl phenol ethoxylate, analogs or derivatives. Octylphenol ethoxylate is also known as polyoxyethylene octyl phenyl ether,4-octylphenol polyethoxylate, Mono 30, TX-100,t-octylphenoxypolyethoxyethanol, octoxynol-9, or the more commonly knowntradename of Triton™ X-100. In alternative embodiments, thepermeabilizing agent comprises Nonoxynol 9, analogs, or derivatives.

In certain embodiments, the permeabilizing agent comprises a cationicpolymer. In some embodiments, the cationic polymer is a mucoadhesive. Insome embodiments, the cationic polymer includes chitosan, chondroitin,chitosan analogs, chondroitin analogs, chitosan derivatives, orchondroitin derivatives.

In some embodiments, the present pharmaceutical composition comprises aclostridial derivative and at least one permeabilizing agent. In oneembodiment, the clostridial derivative is a botulinum toxin. In oneembodiment, the clostridial derivative includes a botulinum toxin. Insome embodiments, the at least one permeabilizing agent compriseschitosan, chitosan analog or chitosan derivative and TX-100, TX-100analogs or TX-100 derivatives. In some embodiments, the presentpharmaceutical composition comprises a botulinum toxin, chitosan,chitosan analog or chitosan derivative and nonoxynol-9, nonoxynol-9analogs or nonoxynol-9 derivatives. In alternative embodiments, theclostridial derivative is a TEM. In alternative embodiments, theformulation comprises a botulinum toxin and a TEM.

In certain embodiments, the permeabilizing agent can comprise chelators,including but not limited to EDTA, EGTA (ethylene glycol tetraaceticacid), cyclohexanediamine tetraacetate (CDTA),hydroxyethylethylenediamine triacetate (HEDTA), diethylenetriaminepentaacetate (DTPA), 1,2-diaminocyclohexane tetraacetate, andhexametaphosphate. These agents preferably are employed as salts,typically sodium salts such as disodium EDTA, trisodium HEDTA, sodiumhexametaphosphate, or any combinations thereof, and the like.

Thus, in one aspect, the present pharmaceutical composition comprises aclostridial derivative, and at least one permeabilizing agent, whereinthe pharmaceutical formulation is suitable for intravesical bladderdelivery. In some embodiments, the clostridial derivative comprises abotulinum toxin and the permeabilizing agent comprises a mucoadhesiveand a surfactant. In some embodiments, the mucoadhesive includeschitosan, chitosan analogs, chitosan derivatives, chondroitin,chondroitin analogs and chondroitin derivatives. In some embodiments,the surfactant includes nonionic surfactants. In one embodiment, thepresent pharmaceutical composition comprises a botulinum toxin type A,chitosan, chitosan analogs or chitosan derivatives and TX-100, TX-100analogs or TX-100 derivatives. In some embodiments, the surfactantcomprises nonoxynol-9, nonoxynol-9 analogs or nonoxynol-9 derivatives.In alternative embodiments, the clostridial derivative is a TEM.

In some embodiments, the permeabilizing agent is present in apermeation-effective amount. In one embodiment, a permeation effectiveamount refers to an amount effective to substantially increase thepermeability of the bladder wall surface with limited damage to thebladder integrity. In one embodiment, the permeation-effective amountrefers to an amount effective to allow permeation of a therapeuticallyeffective amount of a botulinum neurotoxin through the bladder wall at atherapeutically effective rate. In one embodiment, the permeationeffective amount refers to an amount effective to substantially increasethe permeability of the bladder wall surface for a desired time intervalto a therapeutically effective amount of the toxin at a therapeuticallyeffective rate without irreversibly damaging the bladder wall. In someembodiments, the increased permeability is reversible after a desiredtime interval, after which the bladder wall may fully or partiallyrestore its original impermeability or selective permeability. In someembodiments, the bladder wall restores its original impermeability orselective permeability after a time interval ranging from about 1 hourto about 24 hours after intravesical instillation. In some embodiments,the time interval ranges from about 3 hours to about 18 hours. In someembodiments, the time interval ranges from about 4 hours to about 12hours. The recovery rate whereby the bladder wall restores its originalselective permeability or impermeability can be influenced by thecharacteristics of the permeabilizing agent selected, the amount, andthe characteristics of the permeation surface, the exposure time and theenvironment surrounding the permeation surface. In one embodiment, thebladder integrity following administration of the present pharmaceuticalcomposition can be evaluated by the extent of immune response, such asthe presence of specific immune cells.

The permeation-effective amount varies depending on several factors,including but not limited to the characteristics of the clostridialderivative, the characteristics of the permeation surface (e.g. thebladder wall), the type of permeabilizing agent, and the environmentsurrounding the permeation surface.

In some embodiments, the permeation-effective amount of thepermeabilizing agent ranges from about 0.005% to about 10% (w/v), morepreferably from about 0.025% to about 5% (w/v), and most preferably fromabout 0.05% to about 0.5% (w/v). In some embodiments, the presentpharmaceutical formulation comprises a permeation-effective amount ofTriton™ X-100, Triton™ X-100 analogs or derivatives from 0.005% to about10% (w/v), more preferably from about 0.025% to about 5% (w/v), and mostpreferably from about 0.1% to about 0.5% (w/v). In one specificembodiment, the permeative effective amount of Triton™ X-100 is about0.1% (w/v).

In some embodiments, the permeabilizing agent is used in combinationwith a mucoadhesive. In some embodiments, the mucoadhesive is a cationicpolymer. In some embodiments, the mucoadhesive includes chitosan,chitosan analogs, chitosan derivatives, chondroitin, chondroitin analogsor chondroitin derivatives. In some embodiments, thepermeation-effective amount of the mucoadhesive ranges from about 0.005%to 10% (w/v), more preferably from about 0.02% to about 5% (w/v), andmost preferably from about 0.05% to about 2% (w/v). In one embodiment,the permeation-effective amount of chitosan, chitosan analogs orchitosan derivatives is about 1% (w/v). In some embodiments, theformulation comprises: (1) a botulinum toxin, (2) Triton™ X-100, Triton™X-100 analog, Triton™X-100 derivatives or mixtures thereof; and (3)chitosan, chitosan analogs, chitosan derivatives, or mixtures thereof.In some embodiments, the formulation comprises: (1) a botulinum toxintype A, (2) Triton™X-100, Triton™X-100 analogs, Triton™X-100derivatives, or mixtures thereof; and (3) chitosan, chitosan analogs,chitosan derivatives, or mixtures thereof. In some embodiments, theformulation comprises about 20 units to about 300 units of botulinumtoxin type A, about 0.5% to about 2% (w/v) chitosan, chitosan analogs,derivatives; or mixtures thereof, and about 0.05% to about 1% (w/v)Triton™ X-100, analogs, derivatives or mixtures thereof. In oneembodiment, the formulation comprises from about 100 or 200 units ofbotulinum toxin type A, about 1% chitosan and about 0.1% (w/v) Triton™X-100.

In some embodiments, the permeabilizing agent is Nonoxynol 9. In someembodiments, the permeation-effective amount of Nonoxynol 9 ranges fromabout 0.005% to about 10% (w/v), more preferably from about 0.025% toabout 5% (w/v), and most preferably from about 0.05% to about 0.5%(w/v). In one specific embodiment, the present pharmaceuticalformulation comprises a permeation-effective amount of Nonoxynol 9 ofabout 0.1% (w/v). In one specific embodiment, the present pharmaceuticalformulation comprises a permeation-effective amount of Nonoxynol 9 ofabout 0.5% (w/v). In some embodiments, nonoxynol 9 is used incombination with a mucoadhesive. In some embodiments, the mucoadhesiveincludes chitosan, chitosan analogs, chitosan derivatives, chondroitin,chondroitin analogs or chondroitin derivatives. In some embodiments, thepermeation-effective amount of the mucoadhesive ranges from about 0.005%to 10% (w/v), more preferably from about 0.02% to about 5% (w/v), andmost preferably from about 0.1% to about 2% (w/v). In some embodiments,the permeation-effective amount of chitosan or chitosan derivativesrange from about 0.005% to 10% (w/v), more preferably from about 0.02%to about 5% (w/v), and most preferably from about 0.1% to about 2%(w/v). In one embodiment, the permeation-effective amount of chitosan orchitosan derivatives is about 1% (w/v).

In some embodiments, the permeabilizing agent can comprise recombinantor native human serum albumin.

In some embodiments, the present composition does not comprise anyanimal-derived proteins. In one embodiment, the present compositioncomprises a botulinum toxin, a TEM, or a recombinant modified toxin (asthe active ingredient) and a suitable polysaccharide, or non-proteinexcipient as a stabilizer or excipient.

Thus, aspects of the present disclosure provide a pharmaceuticalcomposition comprising a clostridial derivative and one or morepermeabilizing agents, wherein the pharmaceutical composition issuitable for intravesical bladder delivery and wherein the one or morepermeabilizing agent is present in a permeation-effective amount, asdisclosed herein. Embodiments of the present composition includetherapeutic agents and/or excipients that will either cause or enhancethe pharmacological effects of the clostridial derivatives.

Excipients can also be added to increase the stability of theformulation, increase the action of permeabilizing agents (example EDTAor other chelating agents) or to increase the retention of theformulation through increased viscoelastic properties that will occurimmediately (example: carboxymethyl cellulose (CMC), hydroxypropylcellulose (HPMC), alginate) or upon change in temperature (example:poloxamer 407), pH (Carbopol P-934, P940) and/or ionic (example: Gelritegellan gum, alginate) environments.

Excipients can also be added to modulate the tonicity and/or pH of urineand skin to increase delivery, stability, bioavailability and/ortherapeutic activity of formulations.

In another aspect, the present disclosure provides a method for making apharmaceutical formulation for suitable for intravesical bladderadministration, the method comprising providing a solution comprising atleast one permeabilizing agent as disclosed herein, adding the solutionto a composition comprising a clostridial derivative as disclosedherein. In some embodiments, the method comprises adding from about 50ml to about 100 ml of the solution to the composition comprising theclostridial derivative. In some embodiments, the clostridial derivativeis a botulinum toxin. In one embodiment, the method comprises addingabout 50 ml to about 100 ml of an aqueous solution comprising about 1%(w/v) chitosan, analogs or derivatives and about 0.1% (w/v)Triton™X-100, analogs or derivatives, to a botulinum toxin type A. Inone embodiment, the method comprises adding a 50 ml aqueous solutioncomprising about 1% (w/v) chitosan and about 0.1% (w/v) Triton™X-100 toa vial containing about 100 Units or 200 Units of a lyophilizedbotulinum toxin type A; and mixing gently to rehydrate the lyophilizedbotulinum toxin type A.

In some embodiments, excipients can also be added to act as carriers ofthe clostridial derivative. In one embodiment, poly-L-lysine is added asa carrier.

A composition disclosed herein is generally administered as apharmaceutical acceptable composition. As used herein, the term“pharmaceutically acceptable” means any molecular entity or compositionthat does not produce an adverse, allergic or other untoward or unwantedreaction when administered to an individual. As used herein, the term“pharmaceutically acceptable composition” is synonymous with“pharmaceutical composition” and means a therapeutically effectiveconcentration of an active ingredient, such as, e.g., any of theClostridial toxins and/or TEMs disclosed herein. A pharmaceuticalcomposition disclosed herein is useful for medical and veterinaryapplications. A pharmaceutical composition may be administered to anindividual alone, or in combination with other supplementary activeingredients, agents, drugs or hormones. The pharmaceutical compositionsmay be manufactured using any of a variety of processes, including,without limitation, conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, andlyophilizing. The pharmaceutical composition can take any of a varietyof forms including, without limitation, a sterile solution, suspension,emulsion, lyophilizate, tablet, pill, pellet, capsule, powder, syrup,elixir or any other dosage form suitable for administration.

The present pharmaceutical composition may optionally include apharmaceutically acceptable carrier that facilitates processing of anactive ingredient into pharmaceutically acceptable compositions. As usedherein, the term “pharmacologically acceptable carrier” is synonymouswith “pharmacological carrier” and means any carrier that hassubstantially no long term or permanent detrimental effect whenadministered and encompasses terms such as “pharmacologically acceptablevehicle, stabilizer, diluent, additive, auxiliary or excipient.” Such acarrier generally is mixed with an active compound, or permitted todilute or enclose the active compound and can be a solid, semi-solid, orliquid agent. It is understood that the active ingredients can besoluble or can be delivered as a suspension in the desired carrier ordiluent. Any of a variety of pharmaceutically acceptable carriers can beused including, without limitation, aqueous media such as, e.g., water,saline, glycine, hyaluronic acid and the like; solid carriers such as,e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin,talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like;solvents; dispersion media; coatings; antibacterial and antifungalagents; isotonic and absorption delaying agents; or any other inactiveingredient. Selection of a pharmacologically acceptable carrier candepend on the mode of administration. Except insofar as anypharmacologically acceptable carrier is incompatible with the activeingredient, its use in pharmaceutically acceptable compositions iscontemplated. Non-limiting examples of specific uses of suchpharmaceutical carriers can be found in PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS (Howard C. Ansel et al., eds., Lippincott Williams& Wilkins Publishers, 7th ed. 1999); REMINGTON: THE SCIENCE AND PRACTICEOF PHARMACY (Alfonso R. Gennaro ed., Lippincott, Williams & Wilkins,20th ed. 2000); GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OFTHERAPEUTICS (Joel G. Hardman et al., eds., McGraw-Hill Professional,10th ed. 2001); and HANDBOOK OF PHARMACEUTICAL EXCIPIENTS (Raymond C.Rowe et al., APhA Publications, 4th edition 2003). These protocols areroutine procedures and any modifications are well within the scope ofone skilled in the art and from the teaching herein.

A pharmaceutical composition disclosed herein can optionally include,without limitation, other pharmaceutically acceptable components (orpharmaceutical components), including, without limitation, buffers,preservatives, tonicity adjusters, salts, antioxidants, osmolalityadjusting agents, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed herein,provided that the resulting preparation is pharmaceutically acceptable.Such buffers include, without limitation, acetate buffers, citratebuffers, phosphate buffers, neutral buffered saline, phosphate bufferedsaline and borate buffers. It is understood that acids or bases can beused to adjust the pH of a composition as needed. Pharmaceuticallyacceptable antioxidants include, without limitation, sodiummetabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole and butylated hydroxytoluene. Useful preservativesinclude, without limitation, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, phenylmercuric nitrate, a stabilizedoxy chloro composition and chelants, such as, e.g., DTPA orDTPA-bisamide, calcium DTPA, and CaNaDTPA-bisamide. Tonicity adjustorsuseful in a pharmaceutical composition include, without limitation,salts such as, e.g., sodium chloride, potassium chloride, mannitol orglycerin and other pharmaceutically acceptable tonicity adjustor. Thepharmaceutical composition may be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. It is understood that these and other substances knownin the art of pharmacology can be included in a pharmaceuticalcomposition. Exemplary pharmaceutical composition comprising aClostridial toxin and a TEM are described in Hunt, et al., AnimalProtein-Free Pharmaceutical Compositions, U.S. Ser. No. 12/331,816; andDasari, et al., Clostridial Toxin Pharmaceutical Compositions,WO/2010/090677, each of which is hereby incorporated by reference in itsentirety.

The choice of suitable pharmaceutically acceptable carriers will dependon the exact nature of the particular formulation desired, e.g., whetherthe present composition is to be formulated into a liquid solution, alyophilized powder to be reconstituted upon use, a suspension solution,nanoparticles, liposomes or microemulsions.

The choice of a suitable pharmaceutically acceptable carrier will alsodepend on the route of administration. Preferably, the carrier isformulated to be suitable for a chosen route of administration.Administration modes of the present pharmaceutical formulation includebut are not limited to injection, needle-free injections (e.g. Bioject,JetTouch), electromotive, transdermal delivery or intravesicalinstillation. In one specific embodiment, the formulation comprises apharmaceutical acceptable carrier for bladder instillation. In oneembodiment, the pharmaceutical carrier comprises poly lysine.

Methods of Treatment

Aspects of the present disclosure provide, in part, a method of treatingmedical conditions using a pharmaceutical composition comprising aclostridial derivative and at least one permeabilizing agent, whereinadministration of the present pharmaceutical composition prevents orreduces a symptom associated with the medical condition being treated.In some embodiments, the administration is by injection. In alternativeembodiments, the administration is transdermal, subcutaneous, ortopical. In yet alternative embodiments, the administration is byintravesical delivery.

In some embodiments, the present disclosure provides methods of treatingdiseases, disorders, conditions, and the like, comprising the step ofadministering a pharmaceutical formulation of the present disclosure toa subject in need thereof in an amount sufficient to produce improvedpatient function. In certain embodiments, the diseases are of aneuromuscular nature, such as, for example, those diseases that affectmuscles and nerve control thereof, such as, for example, overactivebladder, and the like. Certain embodiments relate to the treatment ofpain, such as, for example, treatment of headache pain, or back pain, ormuscle pain, or the like. In certain embodiments, methods of theinvention encompass the treatment of psychological disorders, including,for example, depression, anxiety, and the like.

Treatment of Urological Disorders

In some embodiments, the medical disorder comprises urological bladderdiseases and conditions, including but not limited to overactive bladder(OAB), cystitis, bladder cancer, neurogenic detrusor overactivity (NDO).In an embodiment, the present disclosure also provides methods fortreating a patient suffering from overactive bladder (OAB), such as, forexample, that due to a neurologic condition (NOAB), or idiopathic OAB(IOAB).

Neurogenic bladder dysfunction is a dysfunction that results frominterference with the normal nerve pathways associated with urination.One type of neurogenic bladder dysfunction is overactive (spastic orhyper-reflexive) bladder. An overactive neurogenic bladder ischaracterized by uncontrolled, frequent expulsion of urine from thebladder. There may be reduced bladder capacity and incomplete emptyingof urine. Spastic bladder may be caused by an inability of the detrusormuscle of the bladder to inhibit emptying contractions until areasonable amount of urine has accumulated. Often, a strong urge to voidis experienced when only a small amount of urine is in the bladder. Thepatient may report symptoms of urgency, frequency, nocturia, andincontinence. Another type of neurogenic bladder dysfunction ischaracterized by difficulty in relaxing the urinary sphincter muscle.The sphincter may be spastic. This causes difficulty in emptying thebladder, which can lead to urinary retention and urinary tractinfections. In another type of neurogenic bladder dysfunction, both thedetrusor muscle and the urinary sphincter simultaneously contractresulting in urinary retention. A dysfunction associated withsimultaneous contraction of both the detrusor and the urinary sphincteris called detrusor-external sphincter dyssynergia (DESD). U.S. Pat. Nos.7,449,192; 8,062,807 and 7,968,104, each of which is hereby incorporatedby reference in its entirety, disclose methods for treating a patientwith a neurogenic bladder dysfunction by injecting a therapeuticallyeffective amount of a botulinum toxin, into the bladder wall of thepatient.

Interstitial cystitis (IC) is an incurable, chronic, debilitatingdisease of the urinary bladder that is characterized by bladder pain,chronic pelvic pain, irritative voiding symptoms and sterile urine. InIC, the bladder wall typically shows inflammatory infiltration withmucosal ulceration and scarring which causes smooth muscle contraction,diminished urinary capacity, hematuria and frequent, painful urination.

In some embodiments, pharmaceutical formulations of the presentdisclosure can be administered to the bladder or its vicinity, e.g. thedetrusor, wherein the administration of the formulation reduces the urgeincontinence associated with overactive bladder. In certain embodiments,the dosage can range from about 10 Units to about 200 U per treatment.In some embodiments, the present pharmaceutical formulations can beadministered by intravesical bladder delivery.

Thus, aspects of the present disclosure further provide for a method fortreating a bladder condition in a patient in need thereof, comprising:providing a pharmaceutical composition as disclosed herein, comprising aclostridial derivative, and at least one permeabilizing agent, whereinthe permeabilizing agent is present in an amount effective tosubstantially enhance the permeability of the bladder wall to theclostridial derivative, at a therapeutically effective rate, and withoutirreversibly damaging the bladder wall; and instilling thepharmaceutically composition to the bladder via a catheter; therebytreating the bladder conditions. In one embodiment, the method furthercomprises pre-treating the bladder wall with the pharmaceuticalcomposition prior to the step of instilling.

In some embodiments, the method for treating a bladder condition in apatient comprising providing a solution comprising a permeabilizingagent; adding the solution to a clostridial derivative to form atherapeutic formulation, instilling the therapeutic formulation througha catheter into a patient's bladder. In some embodiments, theclostridial derivative is a botulinum toxin. In alternative embodiments,the clostridial derivative is a TEM. In one embodiment, the clostridialderivative is a botulinum toxin type A. In some embodiments, thepermeabilizing agent is a surfactant. In one embodiment, thepermeabilizing agent is a nonionic surfactant. In some embodiments, thepermeabilizing agent further comprises a cationic polymer. In someembodiments, the cationic polymer is a bioadhesive or mucoadhesive. Insome embodiment, the mucoadhesive comprises a chitosan, chitosan analogor derivative. In some embodiments, the method comprises mixing asolution comprising a nonionic surfactant and a cationic polymer to abotulinum toxin.

In another aspect, the present disclosure provides a method foralleviating the symptoms of Interstitial cystitis (IC) in a patient, themethod comprising: providing a pharmaceutical composition, comprising aclostridial derivative and at least one permeabilizing agent, whereinthe permeabilizing agent is present in an amount effective tosubstantially enhance the permeability of the bladder wall to theclostridial derivative at a therapeutically effective rate, and withoutirreversibly damaging the bladder wall; and instilling thepharmaceutically composition to the bladder via a catheter; therebyalleviating the symptoms of the IC.

In another aspect, the present formulation maximizes the bioavailabilityof a clostridial derivative by preventing or minimizing the adsorptionof the clostridial derivative to catheters, deliver device surfaces(syringe, patch, microneedle, engineered injector (Bioject, etc.),tubing and containers.

In another aspect, the present formulation maximizes the bioavailabilityof the clostridial derivative by enhancing the retention of theclostridial derivative to the skin or inner bladder wall surface,through mucoadhesive interactions.

Other Medical Disorders:

Treatment of Pain

In another embodiment, the present disclosure provides methods fortreating pain comprising the step of administering a pharmaceuticalformulation of the present invention to a subject in need thereof in anamount sufficient to reduce pain. In another embodiment, the patientsuffers from myofascial pain, migraine headache pain, tension headachepain, neuropathic pain, facial pain, lower-back pain, sinus-headachepain, pain associated with temporomandibular joint disease, painassociated with spasticity or cervical dystonia, post-surgical woundpain, or neuralgia. A treatment session can comprise multipletreatments.

In an embodiment, the patient suffers from facial pain. A subjectsuffering from facial pain, for example, receives between about 4 to 40U per treatment of a pharmaceutical formulation of the presentdisclosure. Dosages greater than 40 U per treatment may also beadministered to patients with facial pain to achieve a therapeuticresponse. A treatment session can comprise multiple treatments.

In an embodiment, the patient suffers from myofascial pain. A subjectsuffering from myofascial pain, for example, receives between about 5 to100 U per treatment of a pharmaceutical formulation of the presentinvention. Dosages greater than 100 U per treatment may also beadministered to patients with myofascial pain to achieve a therapeuticresponse. A treatment session can comprise multiple treatments.

In an embodiment, the subject suffers from lower-back pain. A subjectsuffering from lower-back pain, for example, receives between about 15to 150 U per treatment of a pharmaceutical formulation of the presentinvention. Dosages greater than 150 U per treatment may also beadministered to patients with lower-back pain to achieve a therapeuticresponse. A treatment session can comprise multiple treatments.

In an embodiment, the patient suffers from migraine headache pain,including wherein the patient suffers from migraine headaches of 4 hoursor more 15 or more days per month. A subject suffering frommigraine-headache pain, for example, receives between about 0.5 to 200 Uper treatment of a pharmaceutical formulation of the present invention.A treatment session can comprise multiple treatments.

For example, about 0.5 U, about 1.0 U, about 1.5 U, about 2.0 U, about2.5 U, about 3.0 U, about 3.5 U, about 4.0 U, about 4.5 U, about 5.0 U,about 5.5 U, about 6.0 U, about 6.5 U, about 7.0 U, about 7.5 U, about8.0 U, about 8.5 U, about 9.0 U, about 9.5 U, about 10.0 U, about 12 U,about 15 U, about 17 U, about 20 U, about 22 U, about 25 U, about 27 U,about 30 U, about 32 U, about 35 U, about 37 U, about 40 U, about 42 U,about 45 U, about 47 U, or about 50 U per treatment site areadministered to a patient with migraine-headache pain. A patient can betreated at multiple sites, ranging from 2 sites up to 35 sites. In anembodiment, a patient suffering from migraine is a 31 times with 5 U per0.1 mL injection, across the corrugator (2 injections of 5 U each),procerus (1 injection of 5 U), frontalis (4 injections of 5 U each),temporalis (8 injections of 5 U each), occipitalis (6 injections of 5 Ueach), cervical paraspinal (4 injections of 5 U each), and trapezius (6injections of 5 U each) muscles. With the exception of the procerusmuscle which can be injected at the midline, all muscles can, in certainembodiments, be injected bilaterally with half of the injection sites tothe left and half to the right side of the head and neck. Dosagesgreater than 200 U per treatment may also be administered to patientswith migraine-headache pain to achieve a therapeutic response. Atreatment session can comprise multiple treatments. In alternativeembodiments, the pharmaceutical composition is administeredtransdermally or topically.

In an embodiment, the patient suffers from sinus-headache pain. Asubject suffering from sinus-headache pain, for example, receivesbetween about 4 to 40 U per treatment of a pharmaceutical formulation ofthe present invention. In a further example, the subject receivesbetween about 4 U to 40 U per treatment. Dosages greater than 40 U pertreatment may also be administered to patients with sinus headache-painto achieve a therapeutic response. A treatment session can comprisemultiple treatments.

In an embodiment, the patient suffers from tension-headache pain. Asubject suffering from tension-headache pain, for example, receivesbetween about 5 to 50 U per treatment of a pharmaceutical formulation ofthe present invention. In an embodiment, a patient suffering fromtension headache is injected 31 times with 5 U per 0.1 mL injection,across the corrugator (2 injections of 5 U each), procerus (1 injectionof 5 U), frontalis (4 injections of 5 U each), temporalis (8 injectionsof 5 U each), occipitalis (6 injections of 5 U each), cervicalparaspinal (4 injections of 5 U each), and trapezius (6 injections of 5U each) muscles. With the exception of the procerus muscle which can beinjected at the midline, all muscles can, in certain embodiments, beinjected bilaterally with half of the injection sites to the left andhalf to the right side of the head and neck. Dosages greater than 200 Uper treatment may also be administered to patients with tension headachepain to achieve a therapeutic response. A treatment session can comprisemultiple treatments. In alternative embodiments, the pharmaceuticalformulation may be administered topically or transdermally.

In an embodiment, the patient suffers from sinus headache pain or facialpain associated with acute or recurrent chronic sinusitis. For example apharmaceutical formulation of the present invention can be administeredto the nasal mucosa or to the subcutaneous structures overlying thesinuses, wherein the administration of the formulation reduces theheadache and/or facial pain associated with acute recurrent or chronicsinusitis. In further embodiments, any of the pharmaceuticalformulations of the present invention can be administered to the nasalmucosa or to the subcutaneous structures overlying the sinuses, such asover one or more of the sinuses selected from the group consisting of:ethmoid; maxillary; mastoid; frontal; and sphenoid. In anotherembodiment, subcutaneous structures overlying the sinuses lie within oneor more of the areas selected from the group consisting of: forehead;malar; temporal; post auricular; and lip. In embodiments, multipleinjections of 5 U each are administered to treat the sinus headache painor facial pain associated with acute or recurrent chronic sinusitis.

In another embodiment, a patient suffering from sinus headache pain orfacial pain associated with acute or recurrent chronic sinusitis istreated by administering any of the pharmaceutical formulations of thepresent invention to an afflicted area of the patient. In a furtherembodiment, the pharmaceutical formulations disclosed herein areadministered to the projections of a trigeminal nerve innervating asinus.

Patients suffering from sinus headache pain or facial pain associatedwith acute or recurrent chronic sinusitis often exhibit symptomsincluding rhinitis, sinus hypersecretion and/or purulent nasaldischarge. In one embodiment, patients treated with the pharmaceuticalformulations of the present invention exhibit symptoms of sinushypersecretion and purulent nasal discharge.

Embodiments of the present disclosure also provide methods for treatinga patient suffering from sinus headache pain or facial pain associatedwith acute or recurrent chronic sinusitis, wherein the subject suffersfrom neuralgia. In certain embodiments the neuralgia is trigeminalneuralgia. In another embodiment, the neuralgia is: associated withcompressive forces on a sensory nerve; associated with intrinsic nervedamage, demyelinating disease, or a genetic disorder; associated with ametabolic disorder; associated with central neurologic vascular disease;or associated with trauma. In another embodiment of the presentdisclosure, the pain is associated with dental extraction orreconstruction.

EXAMPLES

The following examples illustrate embodiments and aspects of the presentinvention and are not intended to limit the scope of the presentdisclosure.

In the following examples, in addition to a botulinum toxin type A,BOTOX®, a TEM was used as an active pharmaceutical ingredient. Inseveral examples, the TEM used comprised an opioid peptide targetingdomain, or more specifically a nociceptin peptide. To facilitatescreening of permeability enhancing vehicles in the in vitro 3Durothelial model, fluorescently labeled gelatin (100 kDa) was used as asurrogate (hereafter referred to as “surrogate”). The use of afluorescent surrogate with similar molecular weight as the API to bedelivered allowed the evaluation of the effects of exemplarypermeabilizing agents on the bladder tissues by the use of fluorescence.

Example 1

Use of a human uroepithelial culture model to evaluate effect ofexemplary agents on the permeation of human uroepithelial cells to aselected toxin, TEM, or surrogate fluorescent protein.

The effect of exemplary permeabilizing agents were evaluated in a humanuroepithelial culture model as shown in FIG. 3 .

A Human Uroepithelial Culture Model is shown in FIG. 3 . Briefly, normalhuman bladder epithelial cells (CELLNTECH Cat #HBEP.05) were plated at aconcentration of approximately 150,000 viable cells per well on eitherpolyester (PET) or polycarbonate membrane inserts. The inserts wereplaced inside the wells of 24 well tissue culture plates. CELLNTECHCnT-58 media (growth media) was then added to the wells and the cellswere incubated for 2 to 3 days at 37° C. (5% CO₂) until cells wereconfluent. At the end of incubation, growth media was removed andreplaced with CELLNTECH CnT-21 media (differentiation media). Cells werethen allowed to differentiate for 5 to 7 days after which a 2 to 3 cellhuman uroepithelial layer was established. BOTOX®, a TEM, or abiological surrogate (example 100 kDa Oregon Green 488 labeled Gelatinfluorescent gelatin) could then be formulated in test vehicle andapplied to the membrane surface. The molecular weight of the fluorescentsurrogate was chosen to represent a molecular weight close to the TEMand botulinum type A toxin. Typically a 0.1 mL volume of test solutionwas applied to the surface of the membrane inserts in which cells weregrown on. Samples were then placed back into the incubator. At varioustime intervals flow through was collected in saline (example: EaglesBalanced Salt Saline Solution) or culture media (example: Eagle'sMinimal Essential Medium). Typical sample collection intervals rangedfrom 1 to 3 hours. Other intervals (example: 24 hours) were sometimescollected. Samples were assayed after collection by measuring cell basedpotency (cell-based activity assays specific for BoNT/A or TEM), lightchain cleavage of SNAPtide® 520 (HPLC Light Chain assay) or samplefluorescence.

The LC-HPLC assay has been previously described (Terrence Hunt, DavidRupp, Gary Shimizu, Karen Tam, Julia Weidler and Jack Xie.Characterization of SNARE Cleavage Products Generated by FormulatedBotulinum Neurotoxin Type-A Drug Products. Toxins 2010, 2, 2198-2212.The cell-based assay for BoNT/A has been previously described (EsterFernandez-Salas, Joanne Wang, Yanira Molina, Jeremy B. Nelson, BirgitteP.S. Jacky, K. Roger Aoki. “Botulinum Neurotoxin Serotype A SpecificCell-Based Potency Assay to Replace the Mouse Bioassay”. PLoS One (2012)art. no. e49516 and U.S. Pat. No. 8,198,034 & 8,361,789). The cell-basedassay for TEM has been previously described in U.S. patent applicationUS 20100233741. Each of the prior art disclosed herein is herebyincorporated by reference in its entirety.

Briefly, the light chain assay was carried out as follows: For eachsample, a volume of 225 μL was transferred to separate reaction tubes(microcon tubes) containing 225 μL of 2X digestion buffer (see Table Afor 2X digestion buffer composition). The samples were heated at 37° C.for 30 minutes (reduction step). After the completion of the reductionstep, a volume of 25 μL of 200 μM SNAPtide® 520 was added to each tube(equivalent to 10.5 μM SNAPtide® 520). The reaction samples were thenincubated at 30° C. for 20 hours (digestion step). At the completion ofthe digestion step, a volume of 25 μL of 5% trifluoroacetic acid (TFA)was added to each tube to stop the reaction. The contents of each tubewere then transferred to HPLC vials for analysis.

TABLE A 2X Digestion Buffer Composition 2X Digestion Buffer 1 mM ZnCl₂,4 mM DTT^(a), 0.1% TWEEN 20 in 100 mM HEPES, pH 7.4 Substrate 200 μMSNAPtide ® 520 prepared in SWFI^(b) ^(a)Dithiothreitol ^(b)ListBiological Laboratories, Inc., supplied through Calbiochem (Cat# 567333)

The reaction solutions were analyzed via a reversed-phase highperformance liquid chromatography method (RP-HPLC) using a Waters 2695XE Separations Module and a Waters 2475 Multi λ, Fluorescence Detector(see Table B for RP-HPLC parameters). The samples were eluted using agradient program (see table C) with a mobile phase consisting of 0.1%TFA in water (A) and 0.1% TFA in acetonitrile (B). The data werecollected and analyzed via Waters Empower™ Pro Software (WatersCorporation). The peak areas of the fluorescently labeled cleavageproduct (designated as SNAPtide® 529 by List Biological Laboratories,Inc.) obtained for the injected samples were compared.

TABLE B RP-HPLC Parameters Column Waters Symmetry300 ™ C18, 3.5 μm, 4.6× 150 mm (P/N: 186000197) Column Temperature 35° C. Injection Volume 25μL Flow 1 mL/min Detection Excitation λ = 322 nm Emission λ = 420 nm

TABLE C HPLC Gradient Program Time (min) % A % B 0 90 10 5 90 10 13 8515 18 5 95 20 5 95 21 90 10 30 90 10

Briefly, for the cell-based potency assay for BoNT/A SiMa H1 cells(Allergan, human neuroblastoma cell line) were utilized. For the TEMcell-based assay SiMa P33 hORL-1 stable cell line #6 (Allergan, humanneuroblastoma cell line (SiMa P33, Allergan) transfected with humanORL-1 (hORL-1) plasmid (GeneCopoeia, EX-A1076-M-02, Germantown, MD)).SiMa H1 clonal cell line or SiMa P33 hORL-1 stable cell line #6 werecultured in BD Biosciences brand 175 cm² Collagen IV flasks (62405-652,BD Biosciences, San Jose, Calif.) with vented caps. Growth mediaconsisted of RPMI 1640, 0.1 mM Non-Essential Amino-Acids, 10 mM HEPES, 1mM Sodium Pyruvate, 100 U/mL Penicillin, 100 μg/mL Streptomycin, and 10%Fetal Bovine Serum (FBS). All media components were obtained from LifeTechnologies. Differentiation medium consisted of Neurobasal® media, B27supplement (1X), GlutaMax (1X), 100 U/mL Penicillin, and 100 μg/mLStreptomycin.

For the BoNT/A assay, SiMa H1 cells were plated in differentiation mediaat 100,000 cells per well in a 96 well plate for 3 days. Each well wastreated for 24 h with 0.1 mL of the media removed from the bottomchamber from the permeability assay. The cells in 3 rows were alsotreated with BOTOX® resuspended in SWFI at doses from 0 to 300 Units permL for 24 h as a reference using differentiation media as the diluent.For the TEM assay SiMa P33 hORL-1 #6 cells were plated indifferentiation media at 50,000 cells per well in a 96 well plate forovernight. Each well was treated for 24 h with 0.1 mL of the mediaremoved from the bottom chamber from the permeability assay. The cellsin 3 rows were also treated with AGN-214868 (PRT-2623) at doses from 0to 100 nM for 18 h as a reference.

After the treatment, the cells were cultured in differentiation mediafor an additional 3 days. The media was removed and the cells were lysedfor 1 hour with 30 μL of lysis buffer consisting of 0.15 M sodiumchloride, 20 mM Tris pH 7.5, 0.15 M sodium chloride, 1 nM EDTA, 1 mMEGTA, and 1% Triton X-100 in water. The lysates were centrifuged at 4000rpm for 20 min to eliminate debris.

One microliter of the capture purified monoclonal antibody 2E2A6 (AGN)at 45 μg/mL in PBS containing 750 μg/mL of BSA was custom spotted on MSDHigh Bind plates by Meso Scale Discovery (MSD, Gaithersburg, Md., Cat#L11XB-3). The monoclonal antibody 2E2A6 developed by NTP specificallyrecognizes SNAP25197. Plates were blocked with 150 μL Blocking Bufferconsisting of 2% Amersham blocking reagent (GE-Healthcare, Piscataway,N.J., Cat#RPN418V), 10% goat serum (Rockland Inc., Cat #B304), in PBS-T(0.05% TWEEN 20 (BioRad, Hercules, Calif., Cat #161-0781) in PBS(GIBCO-Invitrogen, Cat #14040)) at room temperature for 1 hour, and theblocking buffer was then discarded.

Twenty five microliters of cell lysate were added to each well of thespotted MSD plate and the plate was incubated at 4° C. overnight. Plateswere then washed three times with PBS-T. Twenty-five microliters ofSULFO-TAG NHS-Ester labeled detection pAb anti-SNAP25 antibody (Ab toN-terminus of SNAP25 that recognizes uncleaved and cleaved products,Sigma, St. Louis, Mo., Cat #59684) in 2% of Amersham blocking reagent inPBS-T at 5 μg/mL were added to the bottom corner of wells. Plates weresealed and shaken at room temperature for 1 h. Plates were washed threetimes with PBS-T, and 150 μL of 1× Read Buffer (MSD, Cat #R92TC-1) wereadded per well. Plates were read in the 516000 Image reader (Meso ScaleDiscovery).

To demonstrate that the in-vitro 3D urothelium is impermeable, like thein vivo human urothelium, as is therefore amenable for screening ofpermeability enhancing vehicles, the surrogate fluorescent gelatin insaline was incubated for 1-3 hours in the upper compartment. Very lowlevels of fluorescent gelatin were recovered in the lower compartmentdemonstrating that the in vitro urothelium is impermeable to these highmolecular weight proteins. Conversely, vehicles known to enhancepermeability in vivo, like EDTA and Accutase, should cause the sameeffect in the in vitro urothelium. Accutase is a protease mixture thatis typically used to harvest cells that are sensitive to treatment withtrypsin. Accutase digest the tight junctions of HBEP cells, but it doesnot affect cell viability. An experiment was completed to measure thepenetration of fluorescent gelatin when the in vitro urothelium istreated with Accutase or EDTA. After 5 minutes pretreatment withAccutase, the in vitro urothelium cell layer allowed 13,000% of the RFUthan the untreated control; while the EDTA treated urothelium increased300%. These data show that the in-vitro urothelium is an impermeablecell layer and that the permeability of the cell layer can be reversed.

Example 2

Triton X-100 and Nonoxynol 9 increased rat bladder permeation to abotulinum toxin type A, a TEM, and a fluorescent gelatin surrogate in ahuman uroepithelial culture model. In this example, effects of TritonX-100, Nonoxynol 9 and Poly-L-lysine Hydrobromide in the presence orabsence of EDTA were evaluated.

The purpose of the study was to determine if the permeability of humanuroepithelial cells to a TEM (100 kDa) could be increased if cells arepretreated for 30 minutes with solutions containing variouspermeabilization increasing agents. The following solutions wereevaluated: saline, saline containing 50 mM EDTA, 1% Triton-X100 insaline, 1% Triton-X100 in saline containing 50 mM EDTA, 1% Nonoxynol 9in saline, 1% Nonoxynol 9 in saline containing 50 mM EDTA, 1%Poly-L-lysine Hydrobromide in saline and 1% Poly-L-lysine Hydrobromidein saline containing 50 mM EDTA. A volume of 0.1 mL of each testsolution was applied to the surface of the membrane inserts in whichcells were grown on. Solutions were removed after 30 minutes incubationat 37° C. (5% CO₂). A volume of 0.1 mL saline containing 0.1 mg/mL TEMwas then placed onto each membrane insert. Samples were then incubatedfor an additional 2 hours at 37° C. (5% CO₂). At 2 hours sample flowthrough was measured by the TEM cell based potency assay and by the HPLCLight Chain assay (Light chain cleavage of SNAPtide® 520), as describedabove.

As shown in FIG. 4 , the results indicate that the permeability of humanuroepithelial cells to fluorescent labeled TEM surrogate could beincreased when cells were pretreated with Triton-X100, Nonoxynol 9 orPoly-L-lysine Hydrobromide. This enhancement occurred with or withoutEDTA.

Several other permeability enhancing vehicles were tested with thesurrogate fluorescent gelatin and with TEM molecules. The results aresummarized in Table D below:

TABLE D Vehicle Description Typical Results SDS Anionic surfactantInhibited Protamine sulfate Cationic Inhibited Benzalkonium chlorideCationic No Effect Poloxamer 100 Nonionic surfactant Increase Brij 97(Polyoxyethylene Nonionic surfactant Increase (dose response) (10)oleoyl ether) Brij 98 (Polyoxyethylene Nonionic surfactant Increase(dose response) (20) oleoyl ether) Triton X Nonionic surfactant Increase(dose response) Nonoxynol 9 Nonionic surfactant Increase (dose response)Tween 20 Nonionic surfactant Increase (dose response) Tween 80 Nonionicsurfactant Increase (dose response) Big Chaps Nonionic surfactantIncrease (dose response) Deoxy Big Chaps Nonionic surfactant Increase(dose response) Polyethylene glycol 3350 Glycol No Effect Benzyl alcoholPreservative Slight increase (1% at 2 and 3 hours) Chitosan MucoadhesiveInhibited Diethylene glycol Glycol No Effect monoethyl ether RevancePeptide Cell penetrating No Effect at 1 through Peptide 3 hoursPoly-L-lysine Peptide Increase (dose response) Poly-L-Arginine PeptideInhibited EDTA Chelator Increase

Example 3

Time and agent concentration dependent transport of fluorescent gelatinsurrogate through the human uroepithelial cells. (FIGS. 5A and 5B)

Briefly, in this example, human uroepithelial cells were exposed tovarious vehicles containing Oregon Green 488 fluorescent labeled gelatinat a concentration of 0.1 mg/mL. The test vehicles consisted of thefollowing: saline, 0.00745% Triton-X100 in saline, 0.0149% Triton-X100in saline, 0.0745% Triton-X100 in saline, 0.149% Triton-X100 in saline,0.745% Triton-X100 in saline, 0.00262% Nonoxynol 9 in saline, 0.00524%Nonoxynol 9 in saline, 0.0262% Nonoxynol 9 in saline, 0.0524% Nonoxynol9 in saline, 0.262% Nonoxynol 9 in saline and 0.524% Nonoxynol 9 insaline. Each sample is also expressed as a multiple of the criticalmicelle concentration (CMC) of Triton-X100 or Nonoxynol 9. The CMC canbe defined as the concentration of surfactant above which micelles arespontaneously formed. One hundred microliter volume of each testsolution was applied to the surfaces of the membrane inserts in whichcells were grown on. Cells were incubated at 37° C. (5% CO₂). At 1, 2and 3 hours sample flow through was sampled and measured for relativefluorescence.

As shown in FIGS. 5A and 5B, the results demonstrate that transport isboth concentration and time dependent. Increased transport was seen whenincreasing surfactant concentration and with increased incubation time.Increased transport was observed for each surfactant vehicle above itsCMC.

Example 4

Effects of exemplary permeabilizing agents on the permeability of humanuroepithelial cells to a botulinum toxin complex (FIGS. 6A and 6B)

The purpose of the study was to determine if the permeability of humanuroepithelial cells to BOTOX® (botulinum type A toxin complex) could beincreased if applied in vehicles containing various concentrations ofTriton-X100 (FIG. 6A) or Nonoxynol 9 (FIG. 6B). The following testvehicles were evaluated: sterile water for injection, 0.01% Triton-X100in sterile water for injection, 0.025% Triton-X100 in sterile water forinjection, 0.05% Triton-X100 in sterile water for injection, 0.01%Nonoxynol 9 in sterile water for injection, 0.025% Nonoxynol 9 insterile water for injection and 0.05% Nonoxynol 9 in sterile water forinjection. Because each BOTOX® vial contains 0.9 sodium chloride,sterile water for injection was used to prepare the vehicles in order tomaintain 0.9% sodium chloride solutions after reconstitution (0.1 mLreconstitution volume). Vials of 100 unit BOTOX® were reconstituted with0.1 mL volumes of each vehicle. The resulting solutions contain 1000BOTOX® units per milliliter. A volume of 0.1 mL of each reconstitutedsolution was applied to the surfaces of the membrane inserts in whichcells were grown on. At 1 and 3 hours sample flow through was measuredby the HPLC Light Chain assay (Light chain cleavage of SNAPtide® 520)and the BoNT/A cell-based assay.

The results shown in FIGS. 6A and 6B demonstrate that permeability ofhuman uroepithelial cells to BOTOX® (botulinum type A toxin complex)could be increased when applied in vehicles containing Triton-X100 (FIG.6A), and Nonoxynol 9 (FIG. 6B). Test results demonstrate thatpermeability is both concentration and time dependent. Increasedpermeability was seen when increasing Triton-X100 and Nonoxynol 9concentration and with increased time (1 hour versus 3 hours). Increasedpermeability is reflected in the increases seen in peak area counts(Snaptide® 520 cleavage).

The sample flow through at 1 hour was also analyzed in the BoNT/Acell-based assay. There was a 3 fold increase vs saline in the amount ofBoNT/A that permeated the in vitro urothelium when BOTOX® wasadministered in the presence of 0.05% Nonoxynol 9, and a 3.6 foldincrease vs. saline when 0.05% Triton-X100 was used.

It has been reported that at a pH value of 6 the 900 kDa botulinum typeA toxin complex will remain intact while at a pH value of 8 the complexwill disassociate resulting in free 150 kDa botulinum type A toxin (1).The following test solutions were evaluated: 5 mM potassium phosphatesaline, pH 6.0; 5 mM potassium phosphate saline, pH 8.0; 0.5%Triton-X100 in 5 mM potassium phosphate saline, pH 6.0 and 0.5%Triton-X100 in 5 mM potassium phosphate saline, pH 8.0. The sample flowthrough was assayed in the BoNT/A cell-based assay. There were nodifferences between BOTOX® in pH 6 or 8, and in both cases, the use of0.5% Triton-X100 increased the permeability of active BoNT/A through thein vitro urothelium (FIG. 7 ).

Example 5

In vivo assessment: Effect of exemplary permeabilizing agents wasevaluated in vivo on rat bladder. In this example, effects of TritonX-100 and Nonoxynol 9 were evaluated (FIGS. 8A, 8B, 9A and 9B)

Methods:

BOTOX® and TEM surrogate administration: For instillation of vehiclecontrol, working solutions of BOTOX® (onabotulinumtoxinA; Allergan) andthe surrogate were prepared in either 0.9% saline or 0.5% HSA,respectively. For instillation of the surfactant, working solutions ofBOTOX® (onabotulinumtoxinA; Allergan) and TEM surrogate were prepared in0.9% saline. The urinary bladder wall was instilled with BOTOX® (20 U)or surrogate (250 μg) for 1 hour, under anesthesia. All in vivoprotocols and procedures were approved by the institutional AACUC.

Tissue preparation: Bladder tissue from adult Sprague Dawley rats(220-250 g, n=2-20) were harvested 2-days post-instillation and fixedovernight at 4° C. in Zamboni's fixative. Tissues were thencryoprotected (30% sucrose), hemisected longitudinally, frozen inembedding medium and kept at −80° C. until use.

Immunofluorescence: One tissue block per animal was cryostat-sectioned(14 μm-thick) in the longitudinal plane and slide-mounted. Three slideswere prepared from each block, with three sections on each slide,approximately 140 μm apart. Two slides were processed forimmunofluorescence. Tissue sections were first blocked for non-specificsignal in blocking buffer (1X PBS+0.1% Triton X-100+10% Normal DonkeySerum) and then incubated with combinations of primary antibodies at thedesired concentration in blocking buffer, overnight at 4° C. The primaryantibodies used were as follows: mouseanti-Calcitonin-Gene-Related-Peptide (CGRP; Sigma, C7113, 1:5,000),anti-cleaved SNAP25 (SNAP25197; Allergan, 1:1,000), rabbit anti-synapticvesicle glycoprotein 2C (SV2C; Santa Cruz Biotechnologies, sc-28957,1:800) and rabbit anti-vesicular acetylcholine transporter (VAChT;Sigma, V5387, 1:3,000). Following washes, sections were incubated withsecondary antibodies (Jackson ImmunoResearch) for 2 hours at 4° C. andthen washed again. Slide-mounted sections were cover slipped using Fluormount-G with 1.5 μg/ml DAPI. The third slide was stained withHematoxylin & Eosin (H&E) for anatomical assessment.

Data analysis and quantitation: Images were analyzed and captured oneither a Leica DMLB brightfield microscope, a Nikon E800 fluorescentwide field microscope or a Zeiss LSM-710 confocal microscope usingImage-Pro® (MediaCybernetics), Metamorph® (Molecular Devices) or ZEN(Carl Zeiss) software. Imaris® (Bitplane) software was used forhigh-resolution, 3D qualitative analysis to establish the spatialrelationships of nerve fibers. Nerve fiber-types were identified on thebasis of their morphology and neurochemistry. For the semi-quantitativeanalysis, for each slide, all 3 sections were carefully observed under amicroscope and a score (from 0 to 5) was given for each animal on eitherthe extent of SNAP25197 staining (as shown in FIG. 8A) or on theintegrity of the bladder anatomy (H&E) (as shown in FIG. 8B). An averagescore was calculated for each treatment/formulation and the results arepresented as a bar graph (FIGS. 9A, 9B).

FIG. 8A shows a Semi-quantitative assessment of rat bladder integrityusing an ordinal scoring method of 0-5; ‘0’ being a normal bladder and‘5’ showing severe pathology.

FIG. 8B shows a Semi-quantitative assessment of the extent of SNAP25-197staining corresponding to VAChT staining of parasympathetic nerve fibersin rat bladder using an ordinal scoring method of 0-5; ‘0’ being noSNAP25-197 staining detected and ‘4.5’ showing near complete overlap ofboth biomarkers. A score of ‘5’ was never observed.

FIG. 9A shows the extent of SNAP25-197 staining (blue bars) and bladdertissue damage based on H&E staining (red bars) following surrogate (250μg) bladder instillation along with increasing concentrations ofsurfactant. Values are means±SD for the given number of animals. *P<0.05significantly different from vehicle control by one-way ANOVA followedby Holm-Sidak post-hoc analysis.

FIG. 9B shows the extent of SNAP25-197 staining (blue bars) and bladdertissue damage based on H&E staining (red bars) following surrogate (250μg) or BOTOX® (20 U) instillation along with increasing concentrationsof surfactant into rat bladders from interstitial cystitis model. Valuesare means±SD for the given number of animals. *P<0.05 significantlydifferent from vehicle control by one-way ANOVA followed by Holm-Sidakpost-hoc analysis.

Example 6

Effect of Triton-X100 on the permeability of the bladder wall to intact900 kDa botulinum type A complex and free 150 botulinum type A. (FIG. 10)

It has been reported that at a pH value of 6 the 900 kDa botulinum typeA toxin complex will remain intact while at a pH value of 8 the complexwill disassociate resulting in free 150 kDa botulinum type A toxin (1).The following test solutions were evaluated: 5 mM potassium phosphatesaline, pH 6.0; 5 mM potassium phosphate saline, pH 8.0; 0.1%Triton-X100 in 5 mM potassium phosphate saline, pH 6.0 and 0.1%Triton-X100 in 5 mM potassium phosphate saline, pH 8.0. A volume of 2.5mL of each solution was used to reconstitute 100 unit BOTOX® vials. Thisresulted in 40 unit per milliliter solutions. A volume of 0.5 mL of eachreconstituted solution was instilled into rat bladders resulting in theinstillation of 20 units.

FIG. 10 shows the extent of SNAP25-197 staining (blue bars) and bladdertissue damage based on H&E staining (red bars) following BOTOX® (20 U)bladder instillation at either pH 6.0 or pH 8.0 in saline or surfactant.Values are means±SD for the given number of animals. *P<0.05significantly different from vehicle control at given pH by t-testanalysis.

Example 7

Effect of an exemplary permeabilizing agent on human bladder cellmucoadhesive retention. In this example, the exemplary permeabilizingagent is Chitosan. (FIG. 11 )

Chitosan demonstrating human bladder cell mucoadhesive retention

Human bladder cells (CELLNTECH Cat #HBEP.05 were plated at 150,000 cellsper well on PET membrane inserts (24 well) in CELLNTECH CnT-58 media(growth media) for 3 days, then in CELLNTECH CnT-21 (differentiationmedia) for 7 days. Cell layer preconditioned in Saline with 0%, 0.025%,0.05%, 0.1%, 0.2%, 0.4% Chitosan or for 10 minutes with Accutase (mildTrypsin)

Cell layer treated with 100 μL 0.5 mg/mL Oregon Green labeled Gelatin.

Flow Through media was Hanks Balanced Salt Saline Solution.

Flow through media was collected and RFU was measured at 30 min, 1 hour,2 hour, and 3 hour.

The amount remaining on membrane visualized and counted, the results areshown in FIG. 11 . The results show that chitosan increased theretention of gelatin within the in vitro urothelium.

Example 8

Triton X-100 induced increased permeability of the bladder wall wasreversible

Human bladder uroepithelial cells (CELLnTEC Cat #HBEP.05) were plated ata concentration of approximately 150,000 viable cells per well onpolycarbonate membrane inserts. The inserts were placed inside the wellsof 24 well tissue culture plates. CELLnTEC CnT-58 media (growth media)was then added to the wells and the cells were incubated for 2 days at37° C. (5% CO₂) until cells were confluent. At the end of incubation,growth media was removed and replaced with CELLnTEC CnT-21 media(differentiation media). Cells were then allowed to differentiate for 7days after which a 2 to 3 cell human uroepithelial layer wasestablished. Cells were treated for one hour with the followingvehicles: 0.9% saline, 0.1% Triton X-100 in 0.9% saline and 0.5% TritonX-100 in saline. Each vehicle was tested in triplicate. Cells weretreated by applying 0.1 mL volumes of each vehicle to the surfaces ofthe membrane inserts in which cells were grown on. After exposurevehicle solutions were removed from the inserts and differentiationmedia was added to each insert. Cells were then incubated and allowed torecover for either 0, 24, or 48 hours. Note that 0.9% saline (negativecontrol) was only tested at 0 hours. Gelatin permeability assays werethen performed. Gelatin permeability assays were performed by removingmedia from each insert followed by adding 0.1 mL volumes of 0.1 mg/mLOregon Green 488 labeled gelatin, formulated in saline, to each insert.Inserts were placed into wells containing 0.8 mL volumes of Earl'sBalanced Salt Solution. After 1 hour exposure, flow through wascollected and measured for fluorescence using an Envision instrument.Twenty-four hour and forty-eight hour test results were then compared to0 hour data to determine if decreases in gelatin permeability hadoccurred.

It was found that the amount of gelatin that diffused through the invitro urothelium treated with 0.1% Triton X-100 was lower after 24 and48 hours recovery. These results suggest recovery of in vitro humanurothelial permeability after treatment with 0.1% Triton X-100.

Example 9 (FIGS. 12, 13, 14A-14D and 15)

The purpose of this study was to evaluate the effect of botulinum toxintype A, known as BOTOX®, in one of eight vehicle formulationsadministered by instillation into the urinary bladder of female SpragueDawley rats. The effect was examined eight days after administrationwherein efficacy and tolerability were evaluated by immunohistochemistry(IHC) and histopathology, respectively.

Positive controls: Four rats were administered 10 units of BOTOX® insaline by injection into the detrusor muscle.

Negative controls: Formulations containing the vehicle only (as shown inTable 1 without addition of BOTOX®).

Cleaved SNAP 25 was used as a biomarker of BOTOX® activity at synapticterminals and a potential indicator of functionality of the method ofdelivery. In this study it was used to confirm the successful movementof BOTOX® across the urothelium. Synaptophysin expression was used toidentify synaptic terminals and to ensure specificity of cleaved SNAP 25localization. Histopathology was done to assess impact of theformulation on the bladder tissue.

Female rats weighing between 150-200 grams were administered 0.5 mlvehicle formulation or vehicle formulation+30 U Botox® according toTable 1:

TABLE 1 UR13002RS Study Number DP Study Number Vehicle Formulation 1DP13104 0.1% Triton 2 DP13112 0.2% Triton 3 DP13117 0.05% Triton 4DP13121 0.1% Nonoxynol-9^(a) 5 DP13129 1% Chitosan + 0.1% Triton 6DP13130 0.25% HPMC + 0.1% Triton 7 DP13139 0.1% Tyloxapol 8 DP13145 1%Chitosan + 0.1% Nonoxynol-9 ^(a)= only 5 vehicle + BOTOX treatedbladders submitted for evaluation.

Intravesical instillation of the formulation was carried out as follows:rats were anesthetized with isoflurane and the bladder emptied by way offinger tip pressure applied on the lower abdomen. While underanesthesia, a catheter was introduced into the urinary bladder via theurethra. Subsequently, the formulation was slowly administered over acourse of two minutes into the urinary bladder at a dose volume of 0.5ml+0.1 ml (to accommodate for the dead space created by the catheter).The formulation was allowed to dwell in the bladder for 60 minutesbefore anesthesia recovery. The rats were euthanized one week afterinstillation.

Urinary bladders were collected, fixed in 10% formalin and processedusing standard histological techniques. Additional sections wereprepared and processed for fluorescent immunohistochemistry for cleavedSNAP 25 and synaptophysin.

Immunofluorescence: One tissue block per animal was cryostat-sectioned(14 μm-thick) in the longitudinal plane and slide-mounted. Three slideswere prepared from each block, with three sections on each slide,approximately 140 μm apart. Two slides were processed forimmunofluorescence. Tissue sections were first blocked for non-specificsignal in blocking buffer (1X PBS+0.1% TX-100+10% Normal Donkey Serum)and then incubated with combinations of primary antibodies at thedesired concentration in blocking buffer, overnight at 4° C. The primaryantibodies used were as follows: mouseanti-Calcitonin-Gene-Related-Peptide (CGRP; Sigma, C7113, 1:5,000),anti-cleaved SNAP25 (SNAP25197; Allergan, 1:1,000), rabbit anti-synapticvesicle glycoprotein 2C (SV2C; Santa Cruz Biotechnologies, sc-28957,1:800) and rabbit anti-vesicular acetylcholine transporter (VAChT;Sigma, V5387, 1:3,000). Following washes, sections were incubated withsecondary antibodies (Jackson ImmunoResearch) for 2 hr at 4° C. and thenwashed again. Slide-mounted sections were coversliped usingFluoromount-G with 1.5 μg/ml DAPI. The third slide was stained withHematoxylin & Eosin (H&E) for anatomical assessment.

Data analysis and quantitation: Images were analyzed and captured oneither a Leica DMLB brightfield microscope, a Nikon E800 fluorescentwidefield microscope or a Zeiss LSM-710 confocal microscope usingImage-Pro® (MediaCybernetics), Metamorph® (Molecular Devices) or ZEN(Carl Zeiss) software. Imaris® (Bitplane) software was used forhigh-resolution, 3D qualitative analysis to establish the spatialrelationships of nerve fibers. Nerve fiber-types were identified on thebasis of their morphology and neurochemistry. For the semi-quantitativeanalysis, for each slide, all 3 sections were carefully observed under amicroscope and a score (from 0 to 4) was given for each animal on eitherthe extent of SNAP25197 staining (as shown in FIGS. 12 and 13 ) or onthe integrity of the bladder anatomy (H&E) (as shown in FIGS. 14A-D). Anaverage score was calculated for each treatment/formulation and theresults are partly presented in Table 2 and FIG. 15 .

The formulations were evaluated based on the following:

-   -   1. Histological changes in the bladder wall; wherein a diagnosis        of “spindle cell infiltrate” was made for bladders in which an        increased number of fusiform cells of indeterminate origin were        found infiltrating the lamina propria (FIGS. 14B, 14C and 14D);        as a reactive change, these were considered undesirable as a        potential precursor to fibrosis; and    -   2. Cleaved SNAP 25 score.

Summary of results: The results are summarized in Table 2.

TABLE 2 infiltrates/ UR13002RS1/ Rat inflammation: spindle cell cleavedDP13104 # Treatment WNL lamina propria infiltrates edema other SNAP 25150 1 0.1% Triton + BTX x 2 (30 U) Instillation 250 2 0.1% Triton + BTXmin mononuclear 0 (30 U) Instillation infiltrates 350 3 0.1% Triton +BTX min mixed cell min, 3 (30 U) Instillation infiltrates lamina propria450 4 0.1% Triton + BTX x 1 (30 U) Instillation 550 5 0.1% Triton + BTXx 0 (30 U) Instillation 650 6 0.1% Triton + BTX x 2 (30 U) Instillation750 7 0.1% Triton min mononuclear 0 Instillation infiltrates 850 8 0.1%Triton x 0 Instillation UR13002RS2/ Rat DP13112 # Treatment 150 10 0.2%Triton min mononuclear 0 Instillation infiltrate 250 11 0.2% Triton +BTX min mixed cell 2 (30 U) Instillation infiltrates 350 12 0.2%Triton + BTX min mononuclear min, 3 (30 U) Instillation cell infiltrateslamina propria 450 13 0.2% Triton + BTX mild mixed cell mild, 2 (30 U)Instillation inflammation lamina propria 550 15 0.2% Triton + BTX mildmixed cell mild, 3 (30 U) Instillation inflammation lamina propria 65016 0.2% Triton + BTX min mixed cell min, 2 (30 U) Instillationinfiltrates lamina propria 750 17 0.2% Triton + BTX x 2 (30 U)Instillation 850 18 0.2% Triton min mononuclear min, 0 Instillation cellinfiltrates lamina propria UR13002RS3/ Rat DP13117 # Treatment 150 190.05% Triton + BTX x 0 (30 U) Instillation 250 20 0.05% Triton minimalmixed cell mild focal lesion 0 Instillation inflammation 350 21 0.05%Triton x 0 Instillation 450 22 0.05% Triton + BTX minimal mixed cellmild 1 (30 U) Instillation inflammation 550 23 0.05% Triton + BTXmoderate mixed cell mod mild 1 (30 U) Instillation inflammation 650 240.05% Triton + BTX moderate mixed cell mod 1 (30 U) Instillationinflammation 750 25 0.05% Triton + BTX x 1 (30 U) Instillation 850 260.05% Triton + BTX moderate mixed cell mod moderate ulceration 2 (30 U)Instillation inflammation infiltrates/ UR13002RS4/ Rat inflammation:spindle cell cleaved DP13121 # Treatment WNL lamina propria infiltratesedema other SNAP 25 150 27 0.1% Nonoxynol-9 minimal mixed cell min, 0inflammation lamina propria 250 29 0.1% Nonoxynol-9 + minimalmononuclear 2 30 U Botox infiltrates 350 30 0.1% Nonoxynol-9 + minimalmixed cell 3 30 U Botox infiltrates 450 31 0.1% Nonoxynol-9 + minimalmononuclear min, patchy 2 30 U Botox infiltrates lamina propria 550 320.1% Nonoxynol-9 + x 2 30 U Botox 650 33 0.1% Nonoxynol-9 + x 3 30 UBotox 750 34 0.1% Nonoxynol-9 mild mixed cell mild, diffuse; 0inflammation lamina propria hemorrhage infiltrates/ UR13002RS8/ Ratinflammation: spindle cell cleaved DP13129 # Treatment WNL laminapropria infiltrates edema other SNAP 25 150 35 1% Chitosan + 0.1% minmononuclear 2 triton + 30 U Botox infiltrate 250 36 1% Chitosan + 0.1%min mononuclear 3.5 triton + 30 U Botox infiltrate 350 37 1% Chitosan +0.1% minimal mixed cell focal 3.5 triton + 30 U Botox inflammation 45038 1% Chitosan + 0.1% min mononuclear epithelium 3 triton + 30 U Botoxinflitrate hyperplasia, min 550 39 1% Chitosan + 0.1% mild mononuclear 3triton + 30 U Botox ifiltrates 650 40 1% Chitosan + 0.1% x 0 triton 75041 1% Chitosan + 0.1% mild mononuclear 4 triton + 30 U Botoxinflammation 850 42 1% Chitosan + 0.1% minimal mixed cell 0 tritoninflammation/ mononuclear iniltrate infiltrates/ UR13002RS6/ Ratinflammation: spindle cell cleaved DP130130 # Treatment WNL laminapropria infiltrates edema other SNAP 25 150 43 0.25% HPMC + 0.1% x 3Triton + BTX (30 U) Instillation 250 44 0.25% HPMC + 0.1% mod mixed cellmild mild 2 Triton + BTX (30 U) inflammation Instillation 350 45 0.25%HPMC + 0.1% mod mixed cell mild mild mild 0 Triton inflammationhemorrhage 450 46 0.25% HPMC + 0.1% x 2 Triton + BTX (30 U) Instillation550 47 0.25% HPMC + 0.1% mod mixed cell mild mild 1 Triton + BTX (30 U)inflammation Instillation 650 48 0.25% HPMC + 0.1% min mixed cell min 2Triton + BTX (30 U) inflammation Instillation 750 49 0.25% HPMC + 0.1% x2 Triton + BTX (30 U) Instillation 850 50 0.25% HPMC + 0.1% severe mixedcell mild mod large ulcer; 0 Triton inflammation bacteria infiltrates/UR13002RS8/ inflammation: spindle cell cleaved DP13145 Rat# TreatmentWNL lamina propria infiltrates edema other SNAP 25 150 59 1% Chitosan +0.1% ZZ min mononuclear 1 Nonoxynol-9 + BTX cell infiltrate (30 U) 25060 1% Chitosan + 0.1% mild mononuclear min 1 Nonoxynol-9 + BTXinfiltrate (30 U) 350 61 1% Chitosan + 0.1% mild mononuclear mindegranulated 2 Nonoxynol-9 + BTX inflammation cells (30 U) 450 62 1%Chitosan + 0.1% x 0 Nonoxynol-9 550 63 1% Chitosan + 0.1% mild mixedcell mild 1 Nonoxynol-9 + BTX inflammation (30 U) 650 64 1% Chitosan +0.1% min mononuclear 2 Nonoxynol-9 + BTX cell infiltrate (30 U) 750 651% Chitosan + 0.1% x 0 Nonoxynol-9 850 66 1% Chitosan + 0.1% mild mixedcell mild 3 Nonoxynol-9 + BTX inflammation (30 U) infiltrates/UR13002RS7/ Rat inflammation: spindle cell cleaved DP13139 # TreatmentWNL lamina propria infiltrates edema other SNAP 25 150 51 0.1%Tyloxapol + x 1 30 U Botox 250 52 0.1% Tyloxapol + x 0 30 U Botox 350 530.1% Tyloxapol + x 0 30 U Botox 450 54 0.1% Tyloxapol + x 0 30 U Botox550 55 0.1% Tyloxapol + x 0 30 U Botox 650 56 0.1% Tyloxapol + x 0 30 UBotox 750 57 0.1% Tyloxapol x 0 850 58 0.1% Tyloxapol mod mixed cell modulcer 0 950 1 0.9% saline + min mixed 4 10 U Botox inflammation, 951 20.9% saline + min mixed 2 10 U Botox inflammation, 952 3 0.9% saline + x3 10 U Botox 953 4 0.9% saline + mod 1 10 U Botox granulomatous

Test formulations: Positive immunohistochemistry scores were observed inall samples post intravesical instillation of BOTOX®, except for theformulations of 0.1% (w/v) Tyloxapol. FIG. 15 shows the IHC scores ofsome exemplary formulations.

Example 10

Treatment of Overactive Bladder

This example describes treatment of patients with hyper reflexivebladder due to neurogenic or idiopathic bladder dysfunction.

Several patients with hyper reflexive bladders symptoms (bladderinfection, incontinence, and urge incontinence) due to neurogenic oridiopathic bladder dysfunction are treated by bladder instillation. Apharmaceutical composition comprising about 100 Units of BOTOX®, 0.1%(w/v) Triton™ X-100 and 1% (w/v) chitosan. The pharmaceuticalcomposition is instilled to the bladder of the patients while underlight sedation. A significant increase in mean maximum bladder capacityand a significant decrease in mean maximum detrusor voiding pressure areobserved 7 days post treatment.

Many alterations and modifications may be made by those having ordinaryskill in the art, without departing from the spirit and scope of thedisclosure. Therefore, it must be understood that the describedembodiments have been set forth only for the purposes of examples, andthat the embodiments should not be taken as limiting the scope of thefollowing claims. The following claims are, therefore, to be read toinclude not only the combination of elements which are literally setforth, but all equivalent elements for performing substantially the samefunction in substantially the same way to obtain substantially the sameresult. The claims are thus to be understood to include those that havebeen described above, those that are conceptually equivalent, and thosethat incorporate the ideas of the disclosure.

We claim:
 1. A pharmaceutical composition comprising a therapeuticallyeffective amount of a clostridial derivative and at least onepermeabilizing agent, wherein: the at least one permeabilizing agent ispresent in an amount effective to substantially and reversibly increasethe permeability of the bladder wall to the clostridial derivative;wherein the clostridial derivative is a botulinum toxin; and wherein theat least one permeabilizing agent comprises a surfactant; and whereinthe surfactant is present in an amount from about 0.005% to about 10%(w/v); and wherein the surfactant is a non-ionic surfactant. themucoadhesive comprises chitosan; and the surfactant comprisesoctoxynol-9.
 2. The pharmaceutical composition of claim 1, wherein theat least one permeabilizing agent further comprises a muchoadhesive. 3.The pharmaceutical composition of claim 1, wherein the at least onepermeabilizing agent comprises octoxynol-9.
 4. The pharmaceuticalcomposition of claim 2, wherein the mucoadhesive comprises chitosan. 5.The pharmaceutical composition of claim 4, wherein the mucoadhesive ispresent in an amount ranging from about 0.01% to about 5% (w/v).
 6. Thepharmaceutical composition of claim 5, wherein the mucoadhesive ispresent in an amount of about 1% (w/v).
 7. The pharmaceuticalcomposition of claim 1, wherein the clostridial derivative is abotulinum toxin type A.
 8. The pharmaceutical composition of claim 3,wherein the surfactant is present in an amount of 0.1% (w/v).
 9. Amethod of treating a patient with a neurogenic bladder dysfunction in apatient in need thereof, comprising intravesically instilling to thebladder wall of the patient a pharmaceutical composition, wherein: thepharmaceutical composition comprising comprises a therapeuticallyeffective amount of a clostridial derivative and at least onepermeabilizing agent present in an amount effective to substantiallyincrease the permeability of the bladder wall to the botulinumneurotoxin at a therapeutically effective rate; wherein the clostridialderivative is a botulinum toxin; and wherein the at least onepermeabilizing agent comprises a non-ionic surfactant; and wherein thenon-ionic surfactant is present in an amount ranging from about 0.05% toabout 10% (w/v).
 10. The method of claim 9, wherein at least onepermeabilizing agent further comprises a muchoadhesive.
 11. The methodof claim 9, wherein the at least one permeabilizing agent comprisesoctoxynol-9.
 12. The method of claim 9, wherein the mucoadhesivecomprises chitosan.
 13. The method of claim 9, wherein the mucoadhesiveis present in an amount ranging from about 0.01% to about 5% (w/v). 14.The method of claim 9, wherein the mucoadhesive is present in an amountof about 1% (w/v).
 15. The method of claim 9, wherein the clostridialderivative is a botulinum toxin type A.
 16. The method of claim 11,wherein the surfactant is present in an amount of 0.1% (w/v).
 17. Apharmaceutical composition comprising a therapeutically effective amountof a clostridial derivative and at least one permeabilizing agent,wherein: the at least one permeabilizing agent is present in an amountof 0.005-10% (w/v) to substantially and reversibly increase thepermeability of the bladder wall to the clostridial derivative; whereinthe clostridial derivative is a botulinum toxin; wherein the at leastone permeabilizing agent comprises a non-ionic surfactant and amucoadhesive,
 18. The pharmaceutical composition of claim 17, whereinthe surfactant is a non-ionic surfactant octoxynol-9 and themucoadhesive is a cationic polymer chitosan.
 19. The composition ofclaim 18, wherein the surfactant is present at about 0.1% (w/v) and themucoadhesive is present at about 1% (w/v).
 20. A method of treating aneurogenic bladder dysfunction in a patient in need thereof, comprisingintravesically instilling to the bladder wall of the patient apharmaceutical composition of claim 17.